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Antibody discovery and structural studies of the viral oncogene ORF74
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Antibody discovery and structural studies of the viral oncogene ORF74
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1
ANTIBODY DISCOVERY AND
STRUCTURAL STUDIES OF
THE VIRAL ONCOGENE ORF74
A dissertation presented to
FACULTY OF THE USC GRADUATE SCHOOL
in candidacy for the degree of Doctor of Philosophy
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)
James Bowman III
Thesis Advisor Dr. Jae U. Jung | August 2019
2
Table of Contents
ACKNOWLEDGEMENTS ......................................................................................................................................... 4
CHAPTER 1 INTRODUCTION .................................................................................................................................. 5
ONCOGENIC VIRUSES ..................................................................................................................................................... 6
KSHV LIFECYCLE AND ASSOCIATED MALIGNANCIES ............................................................................................................... 7
MOLECULAR BASIS OF KSHV MEDIATED ONCOGENESIS ......................................................................................................... 8
G PROTEIN-COUPLED RECEPTOR SIGNALING AND FUNCTION ................................................................................................. 10
PURIFICATION AND STRUCTURAL BIOLOGY OF G PROTEIN-COUPLED RECEPTORS ....................................................................... 13
CAMELID SINGLE CHAIN “NANOBODIES” ........................................................................................................................... 15
SUMMARY OF THESIS ................................................................................................................................................... 16
CHAPTER 2 PROTEIN ENGINEERING AND LARGE-SCALE EXPRESSION .................................................................. 18
INTRODUCTION ........................................................................................................................................................... 19
RATIONAL MUTAGENESIS OF ORF74 TO IDENTIFY STABILIZING MUTATIONS ............................................................................. 20
SCREENING FUSION PARTNERS FOR PROTEIN STABILITY ........................................................................................................ 22
SYSTEMATIC MUTAGENESIS TO GENERATE COVALENT LIGAND COMPLEX .................................................................................. 24
PURIFICATION OF COVALENT RECEPTOR-LIGAND COMPLEX ................................................................................................... 30
OPTIMIZING EXPRESSION IN MAMMALIAN CELLS ................................................................................................................ 33
EXPRESSION OF LIGAND-RECEPTOR COMPLEX IN INSECT CELLS ............................................................................................... 39
CRYSTALLIZATION TRIALS ............................................................................................................................................... 41
DESCRIPTION OF EXPRESSION CONSTRUCTS ...................................................................................................................... 46
Table 5 Constructs for transient transfection of receptor in mammalian cells .................................................. 46
Table 6 Constructs for transient transfection of ligands in mammalian cells .................................................... 51
Table 7 Constructs for baculovirus mediated expression of receptor and ligand in mammalian cells .............. 53
Table 8 Constructs for baculovirus mediated expression of receptor and ligand in insect cells ........................ 54
CHAPTER 3 IDENTIFICATION OF NANOBODIES THAT BIND ORF74 ....................................................................... 55
INTRODUCTION ........................................................................................................................................................... 56
YEAST NANOBODY SURFACE DISPLAY ................................................................................................................................ 56
SELECTION STRATEGY ................................................................................................................................................... 57
SUMMARY OF ORF74 NANOBODY SCREENING .................................................................................................................. 59
BIOCHEMICAL CHARACTERIZATION OF NANOBODIES ............................................................................................................ 60
SUMMARY ................................................................................................................................................................. 61
CHAPTER 4 CONCLUSIONS .................................................................................................................................. 62
SUMMARY AND FUTURE PLANS FOR STRUCTURAL AND ANTIBODY STUDIES ............................................................................... 63
CHAPTER 5 MATERIALS AND METHODS .............................................................................................................. 64
CELLS ........................................................................................................................................................................ 65
TRANSFECTION ........................................................................................................................................................... 65
IMMUNOBLOT ............................................................................................................................................................ 66
RECOMBINANT BACMID PRODUCTION .............................................................................................................................. 67
PRODUCTION AND TITERING OF BACULOVIRUS ................................................................................................................... 68
FLOW CYTOMETRY ....................................................................................................................................................... 69
PROTEIN EXPRESSION BY BACULOVIRUS AND PURIFICATION .................................................................................................. 70
YEAST NANOBODY SELECTION ........................................................................................................................................ 72
NANOBODY PURIFICATION ............................................................................................................................................ 73
ANALYTICAL SIZE EXCLUSION CHOMATOGRAPHY AND GEL FILTRATION ................................................................................... 74
THERMALSTABILITY ASSAY ............................................................................................................................................. 74
RECONSTITUTION OF RECEPTOR IN LIPID CUBIC PHASE ......................................................................................................... 75
CRYSTALLIZATION TRIALS .............................................................................................................................................. 75
3
CLONING AND PRIMERS ................................................................................................................................................ 78
REFERENCES ....................................................................................................................................................... 91
ADDENDUM: POST-TRANSLATIONAL MODIFICATION OF HOIP BLOCKS TLR4-MEDIATED LINEAR UBIQUITIN CHAIN
FORMATION ....................................................................................................................................................... 95
HIGHLIGHTS ............................................................................................................................................................... 96
ABSTRACT .................................................................................................................................................................. 96
IMPORTANCE .............................................................................................................................................................. 97
INTRODUCTION ........................................................................................................................................................... 98
RESULTS .................................................................................................................................................................. 100
DISCUSSION ............................................................................................................................................................. 107
MATERIALS AND METHODS ......................................................................................................................................... 110
REFERENCES ............................................................................................................................................................. 116
FIGURE LEGENDS AND FIGURES .................................................................................................................................... 120
4
Acknowledgements
Individual Acknowledgement
Personal Mom and Dad You taught me perseverance and supported my
fascination with the natural world
Katie and Austin For your shared love of reading and learning
Christina For your loving support and devotion
Scientific
Professor Jae Jung Your offer of a technician job 8 years ago is what
made this document possible, thank you for your
support and tremendous enthusiasm for taking on
challenging projects
Mary Rodgers Thank you for teaching me the fundamental principles
of laboratory science, you provided an example of
being committed to excellence that I can only try to
emulate
Professor Chengyu
Liang
For being the chair of and sole remaining committee
member, I thank you for your critical feedback
Ji In Park Your outstanding work as summer student was critical
for generating a covalent receptor/ligand complex
Ming Lee You taught me the fundamentals of membrane protein
purification and provided valuable guidance
Andrii Ishchenko Your guidance was essential for learning to set up
crystallization trials in the lipid cubic phase
Jeffrey Velasquez Instruction on recombinant bacmid techniques
Kelly Villers Producing and titering baculovirus
Martin Audet Guidance on crystallization trials
Benjamin Stauch Guidance on protein labeling and crystallization
Professor Ray Stevens For providing access to state of the art facilities and
expertise that made this project possible
Professor Vadim
Cherezov
For valuable feedback and guidance on crystallization
trials
Lin-Chun Chang For teaching me yeast culture and amplifying the
nanobody library
Younho Choi I greatly appreciate your discussions and generosity
I-Chueh Huang For agreeing to evaluate my dissertation and defense
All Jung lab members For critical discussions
5
Chapter 1 Introduction
6
Oncogenic Viruses
The idea that disease could be transferred between organisms by objects too small to
be seen with a light microscope was proposed by Jacob Henle in 1840. The term for
these objects would eventually come from the Latin word “virus” which means slimy
liquid or poison. However, it took nearly 100 years from when Henle first proposed the
existence of small infectious agents until concrete evidence for the existence of a viral
particle which mediated transfer of disease was obtained. Electron micrographs of a
solution containing material that caused mosaic disease of tobacco, revealed the first
image and structure of a virion, tobacco mosaic virus in 1939. This work showed the
existence of an infectious viral unit which could transmit disease between organisms.
Remarkably, some of the viruses discovered at this time where shown to be responsible
for an infectious basis for animal cancers including leukemia (V. Ellerman, 1909) and
solid tumors (Rous, 1910). These findings established the concept that animal cancers
could have a viral origin, which provided rationale for investigating a viral basis for
specific human cancers (Burkitt, 1962).
Since the discovery of the first human oncogenic virus in the 1960s (M. A. Epstein,
1964) five additional human tumor viruses have been discovered. Viral infection is now
implicated in 15% of human cancers worldwide (V. Bergonzini, 2010). Specifically,
human papilloma virus, Epstein-Barr virus, human t-lymphotropic virus, Merkel cell
polyomavirus, hepatitis c virus, hepatitis b virus, and Kaposi’s Sarcoma associated
herpesvirus (KSHV) are all demonstrated to have oncogenic potential. The infectious
basis of the cancers derived from these viruses has made the research into their
7
mechanisms of pathogenesis of great public health importance. FDA approved
therapies for hepatitis c and virus and a vaccine for human papilloma virus have
dramatically reduced the number of cancers derived from these two infections (Mesri,
Feitelson, & Munger, Human Viral Oncogenesis: A Cancer Hallmarks Analysis, 2014).
Despite the great progress in understanding the pathogenesis of these two infectious
agents, specific therapies for the other viruses are lacking. We are specifically
interested in KSHV, which in the clinic, currently has no standard of care leading
doctors to rely on a variety of chemotherapeutic and surgical treatments that often have
significant side effects.
KSHV lifecycle and associated malignancies
The clinical symptoms of Kaposi’s Sarcoma (KS), proliferation of abnormal and leaky
blood vessels on the skin were first described by the physician Moritz Kaposi in 1872
(Kaposi, 1872) This form of KS is referred to as classic KS and is prevalent in elderly
Mediterranean males. In patients with untreated acquired immunodeficiency syndrome
(AIDS), it is the most prevalent cancer and also one of the first symptoms that leads
patients find out they are infected with human immunodeficiency virus (HIV) (Mesri,
Cesarman, & Boshoff, Kaposi's sarcoma and its associated herpesvirus, 2010). With the
advent of effective HIV therapy, KS incidence in developed countries has decreased.
However, there is still significant occurrence in children in sub-Saharan Africa (Ziegler,
1993) and the therapeutic options are limited as there is no virus specific therapy
available.
8
KS lesions occur in the dermal layer of the skin, and in severe cases can also involve
the lungs and gastrointestinal tract (Pantanowitz & Dezube, 2008). The major target cell
for infection in these lesions appears to be endothelial cells, which undergo
transformation upon infection with KSHV (Wang, et al., 2004). Upon infecting cells, the
KSHV virion particle, which is composed of a linear ~140kb double-stranded DNA
genome, surrounded by a capsid, tegument, and lipid bilayer, delivers its genome to the
nucleus where it circularizes, and is maintained as a non-integrated episome (Renne,
Lagunoff, Zhong, & Ganem, 1996). Upon infection the virus enters latency where gene
expression is limited to a minimal set of genes in a ~10kb region of the genome which
ensure maintenance of the viral episome (Dittmer, et al., 1998). Through mechanisms
that are unclear in vivo, but can be induced through multiple pathways in vitro, including
with the use of histone deacetylase inhibitors, virally infected cells undergo lytic
reactivation. During this phase transcription of the viral genome becomes widespread in
order to facilitate viral replication and immune evasion. Ultimately lytic reactivation leads
to cell death and propagates the viral infection (Yogev & Boshoff, 2013).
Molecular basis of KSHV mediated oncogenesis
The discovery of KSHV as the etiological agent of multiple human diseases led to the
investigation of the specific viral factors responsible for oncogenesis. Expression of
multiple different genes was shown to induce cellular transformation, and thus far nine
putative oncogenes including: K1, vIL-6, vBCL-2, vIRF1, Kaposin, vFlip, vCyclin, LANA,
9
and ORF74, have been identified
(Montaner, et al., 2006) (Mesri,
Cesarman, & Boshoff, Kaposi's
sarcoma and its associated
herpesvirus, 2010). However,
expression of each of these
genes individually in endothelial
cells which were then
transplanted into mice
demonstrated that only ORF74
expression was sufficient to
induce KS like tumors. ORF74 was first discovered as a viral homolog of a seven-
transmembrane g-protein coupled receptor (GPCR) and is sometimes referred to as
vGPCR (Bais, et al., 1988) (Arvanitakis, Geras-Raaka, Varma, Gershengorn, &
Cesarman, 1997).
Expression of ORF74 in cells induces multiple different signaling pathways which lead
to cell proliferation, cell survival, angiogenesis, and inflammation (Montaner, Kufareva,
Abagyan, & Gutkind, 2013). In simplified terms the mechanism of ORF74 mediated
oncogenesis occurs through three primary mechanisms. Specifically, activation of
ORF74 promotes survival of ORF74 expressing cells by activating AKT and preventing
apoptosis (Montaner, Sodhi, Pece, Mesri, & Gutkind, 2001). ORF74 also activates pro-
inflammatory signaling which leads to Nf-κB mediated production of chemokines such
Figure 1 Mechanism of ORF74 mediated
transformation
PI3K!
AKT (1) Survival
NF-κB
(2) CXCL1
(3) VEGF
ORF74
10
as CXCL1 which further stimulate ORF74 activity as well as soluble factors that can
transform bystander cells (Martin, Galisteo, Ji, Montaner, & Gutkin, 2008). The ability of
ORF74 to transform bystander cells helps reconcile the fact that in animal models of KS
only a small percentage of tumor cells are ORF74 positive (Montaner, Sodhi, Molinolo,
Bugge, & Sawa, 2003). Further, depletion of that small set of ORF74 expressing cells
can completely reverse disease progression in an animal model suggesting that only a
few cells which express the receptor are necessary and sufficient for tumor growth.
The major caveat for the role of ORF74 in human KS is that it is a lytic gene which
should only be expressed in cells undergoing host mRNA shut off and destined to die
(Ganem, 2010). However, data from clinical trials of patients treated with ganciclovir,
which blocks lytic replication, showed a reduction in KSHV levels, suggesting that lytic
replication is in fact required to maintain KSHV infection (Martin, et al., 1999).
Additionally, cells infected with KSHV in a mouse model actually had a survival
advantage over noninfected cells, and importantly, this was dependent on ORF74
expression (Mutlu, et al., 2007). Taken together, this evidence suggests that the potent
signaling ability of ORF74 could be sufficient to initiate oncogenesis even when its
expression is limited. This along with studies showing that depletion of receptor positive
cells blocks tumor growth, indicate that ORF74 is a potential therapeutic target.
G protein-coupled receptor signaling and function
ORF74 is a seven-transmembrane G-protein coupled receptor (GPCR). In humans, the
over 800 GPCR genes comprise the largest gene family in the genome (Frederiksson,
11
Lagerstrom, Lundin, & Schioth, 2003). Functioning as information conduits between the
extracellular and intracellular space by spanning cell membranes, GPCRs are critical
regulators of nearly every physiological process from perception of light, taste, pain,
blood pressure, inflammation, metabolism and cellular trafficking. The critical function
and location of these proteins on the cell surface helps explain why over 30% of FDA
approved drugs directly target the protein products of these genes.
GPCRs are composed of an extracellular ligand binding region, a transmembrane
region, and a cytosolic effector protein binding region. They are able to relay a signal
across the membrane when a ligand binds to the extracellular domain and induces a
conformational change across the transmembrane region which allows binding of a
downstream effector molecule (Weis & Kobilka, 2018). This mechanism requires that
GPCRs have inherent structural flexibility, and depending on the nature of the ligand
bound, the receptor may adopt a conformation which either promotes or inhibits
signaling. Depending on the structural conformation of the cytoplasmic region of the
receptor, two different downstream effector pathways can be activated. The G protein
pathway relies on binding of the heterotrimeric G⍺β! complex to the receptor (De, De
Lean, Stadel, & Lefkowitz, 1980). The G⍺ subunit then undergoes a conformational
change which triggers the release of GDP and binding of GTP. Once bound to GTP the
G⍺ subunit dissociates from the Gβ! and binds to the enzyme adenylyl cyclase, where it
mediates production of the second messenger cyclic-AMP. The released Gβ! then
activates ion channels on the plasma membrane. Both events amplify the receptor
dependent signaling and activate further downstream effector molecules. There are 4
12
different families and 16 unique G⍺ proteins which provide signaling specificity be their
ability to differentially regulate adenylyl cyclase (Cabrera-Vera, Vanhauwe, Thomas,
Medkova, & Preininger, 2003). The second major pathway for receptor activation is the
arrestin dependent signaling pathway. Upon ligand binding to the receptor a G-protein
receptor kinase phosphorylates the C-terminal tail of the receptor. Phosphorylation
allows arrestin to bind to the receptor and trigger its internalization to turn off the
signaling.
GPCRs are classified into five main families based on phylogenetic analysis, of these
the largest is the class A rhodopsin family which contains the chemokine receptors.
ORF74 is most closely related to the chemokine receptor family of GPCRs due to its
promiscuous binding to chemokine ligands (Rosenkilde & Schwartz, 2000). Despite the
fact that it shares a ligand profile most similar to human CXCR2, the sequence
conservation between CXCR2 and ORF74 is 26%. In contrast the sequence identity
between closely related CXCR1 and CXCR2 is 76%. The phylogenetic distance
between ORF74 and human receptors suggests ORF74 has evolved unique functions.
Indeed, sequences that are highly conserved in host receptors and involved in the
inactive to active transition of the receptor are not conserved in ORF74 (Montaner,
Kufareva, Abagyan, & Gutkind, 2013). This is in accordance with one of the most
striking features of ORF74, its high constitutive signaling activity in the absence of any
known ligand. This activity as well as its binding to a chemokine agonist is required for
its oncogenic function (Maussang, Vischer, Leurs, & Smit, 2009). The signaling ability of
ORF74 is not only more potent than host chemokine receptor CXCR1/2, but is also
13
more potent than other viral GPCRs (Casarosa, et al., 2001). The highly unique nature
of ORF74 makes it any interesting target for further characterization by structural
studies which could ultimately facilitate the development of small molecules that inhibit it
for therapeutic intervention in KSHV.
Purification and structural biology of G protein-coupled receptors
Designing new drugs to target GPCRs implicated in disease is enhanced with detailed
structural knowledge of the ligand binding pocket of the receptor. These efforts have
been greatly enhanced since the first structures of a human GPCR were solved in 2007
(Cherezov, et al., 2007). Crystallizing GPCRs first requires the ability to isolate large
quantities of pure, homogenous, stable and functional receptor from cell membranes.
Numerous techniques have been developed in order to address these hurdles (Table 1)
(Munk, et al., 2019)
Table 1 Strategies to facilitate crystallization
Technique Function
Protein
Engineering
N and C terminal truncation Remove disordered region
Glycosylation site removal Increase sample homogeneity
Point mutation Increase expression and stability
Fusion partner Increase expression and stability
Stabilization
outside of
membranes
Nonionic alkyl glucoside
detergents
Mild extraction of receptor from
membrane into micelle, slow off rate
Cholesterol Causes detergent to form bicelle
architecture
14
Ligands Stabilize receptor in specific
conformation
Binding proteins Stabilize receptor in specific
conformation
The ability to purify large quantities of receptor through receptor engineering, as well as
the development of the lipid cubic phase method for crystallizing membrane proteins
(Caffrey, A comprehensive review of the lipid cubic phase or in meso method for
crystallizing membrane and soluble proteins and complexes, 2015) and high through
put microscale crystallization platforms (Abola, Kuhn, Earnest, & Stevens, 2000) has led
to an explosion of structural information on GPCRs. This has provided important
information for the atomic basis of GPCR activation upon ligand binding that allows
coupling to a heterotrimeric G protein (Rasmussen, et al., 2011). Further this structural
knowledge has accelerated drug discovery and led to the development of molecules
now in clinical trials (Katrich, et al., 2010) (Hanson, et al., 2012). In this thesis I will
describe the utilization of these techniques and platforms to engineer a thermal stable
ORF74 protein which can be purified at milligram scale to be used for antibody
screening and structural studies. However, due to the fact that ORF74 exhibits high
constitutive activity, it presents unique problems from a structural biology standpoint.
To date, 62 unique receptor structures have been solved, and only 22 of those have
been solved in an active state (gpcrbd.org). The active state structures have revealed
how ligand binding in the extracellular pocket triggers an outward movement of
15
transmembrane domain 6 (TM6) of 14Å away from central TM3, thus creating a novel
interaction surface on the cytoplasmic side for the G protein to bind. Crystal structures
of individual receptors in different states have been essential for understanding the
dynamic nature of GPCRs and how specific ligands modulate their activity. What is
apparent from the structural data available, is that the active state of receptors is more
challenging to crystallize, likely due to its relative instability compared to an inactive
conformation. The majority of the active state structures solved required the presence of
a protein bound to the cytoplasmic side of the receptor to stabilize the active
conformation. These proteins have been either heterotrimeric G proteins and camelid
derived single chain antibodies. As a result, it may be required to identify a stabilizing
protein to enable the crystallization of ORF74.
Camelid single chain “nanobodies”
Camelid species naturally produce both conventional heavy and light chain
immunoglobulin molecules and heavy chain only immunoglobulin (Hamers-Casterman,
et al., 1993). The variable region from a
heavy chain only nanobody is
approximately 14 kDa and is referred to
as a nanobody (Figure 2). The These
antibodies have unique features when
compared to conventional antibodies
including their ability to recognize conformation epitopes and bind to buried hydrophobic
regions of antigens. Additionally, they can be expressed and purified from bacteria as
Nanobody
Heavy
Chain
Light Chain
Figure 2 Camelid derived nanobody
16
well as yeast and mammalian cells. Further there is evidence that nanobodies stabilize
biologically relevant conformations of flexible proteins such as GPCRs rather than
inducing artificial states (Smirnova, et al., 2015). As a result, nanobodies have become
valuable tools for structural biology, indeed the structure of the human cytomegalovirus
GPCR was solved in complex with a nanobody (Burg, et al., 2015). In addition to their
use structural biology, nanobodies can be used for in vitro diagnostic assays and are
currently being used in multiple human clinical trials (Saerens, Ghassabeh, &
Muyldermans, 2008). A nanobody that binds to ORF74 could therefore have multiple
potential uses, as a detection agent for biopsies, therapeutic agent to block signaling, or
as a chaperone to promote crystallization of the receptor.
Summary of thesis
The most effective antiviral medicines target virus specific proteins, and the most widely
approved human medicines target GPCRs. Thus, ORF74 has a unique status as both
belonging to a highly druggable protein class and being a virus specific factor. The
KSHV gene ORF74 has been convincingly demonstrated to act as an oncogene which
leads to Kaposi Sarcoma like phenotypes in animal models. Further, eradication of
ORF74 dependent signaling is able to resolve disease, providing proof of principle that
ORF74 is a therapeutic target. Despite the discovery of ORF74 mediated oncogenesis
over 20 years ago, there is an absence of both tools to study ORF74 in human patients
as well as molecules that could target the receptor for therapeutic intervention. Using
mutagenesis, fusion partner screening, and covalent ligand complex formation, I
engineered ORF74 protein to be stable when isolated from cell membranes. I then used
17
this protein to identify three synthetic nanobodies which recognize native ORF74
expressed on cell membranes. Additionally, I used the purified protein for initiating
crystallization trials which are still ongoing. The antibodies I discovered could be used to
investigate the function of ORF74 in human samples which until now has not been
possible. Additionally, the platform I established for purifying milligram quantities of
receptor can provide the foundation for future structural studies and structure-based
drug design.
18
Chapter 2 Protein engineering and large-scale expression
19
Introduction
In order to initiate crystallization trials of a GPCR target milligram quantities of protein
that is greater than 90% pure, homogenous, thermal stable (Tm > 55° C), and highly
concentrated (>25mg/ml) is necessary. Additionally, the receptor should be bound to a
high affinity ligand as only a single receptor thus far has been crystallized in a ligand
free (apo) conformation. Finally, the majority of GPCRs is buried in a membrane
environment and is unable to form polar contacts which could nucleate crystallization.
Thus, a suitable rigid and easily crystallizable protein is typically fused to either the
receptor N-terminus or intracellular loop 3 (ICL3). Collectively these requirements
necessitate the screening of mutations which promote receptor thermalstability, various
receptor ligands, and both fusion partner and fusion junction sites. Multiple different
vectors utilizing different promoters were analyzed as well. The first part of this chapter
will address the engineering of ORF74 according to these guidelines. In total 344
unique constructs were cloned and screened for this project and are detailed at the end
of this chapter.
Further, special care must be taken to ensure that a proper expression host is selected
for purifying the protein. Optimizing the large scale expression of the receptor is the
focus of the second portion of chapter 2. Due to the presence of multiple
transmembrane domains and post translational modification such as glycosylation and
tyrosine sulfation, bacteria are not a suitable expression host for ORF74. Thus we
chose to evaluate expression of the receptor using three different eukaryotic cell lines,
insect Sf9 cells, mammalian 293T cells, and a 293S variant which lacks the enzyme
20
required for producing complex glycosylation. Expression of the receptor was tested
using multiple different methods including transient transfection in suspension and in
adherent cells, baculovirus transduction of Sf9, and baculovirus transduction of 293S
cells. Through construct and expression system screening I identified conditions that
enabled the purification of milligram quantities of pure, homogenous, and stable ORF74
in a covalent complex with a chemokine ligand.
Rational mutagenesis of ORF74 to identify stabilizing mutations
Initial expression and purification of wildtype ORF74 in insect cells produced low levels
of protein, and what protein that was purified was mostly aggregated as determined by
gel filtration analysis and thermalstability assays. Further, small scale expression of WT
receptor was barely detectable by Western blot. To bypass the time-consuming process
of screening mutations using insect cells, which takes 3-4 weeks to validate a construct,
I instead focused on transient expression in mammalian cells which can assess a
construct in 2-3 days. Thus, I first screened for mutations that could increase the
expression of ORF74 when transiently expressed in 293T cells. Initial mutations were
chosen based on multiple different approaches and are summarized in Table 2. First
the sequence of ORF74 was aligned with a receptor that was evolved by random
mutagenesis to be highly stable (Sarkar, et al., 2008). Residues that stabilized that
receptor were used to introduce a corresponding mutation into ORF74. Additional
mutational approaches relied on identification of highly conserved residues in
crystallized receptors and chemokine receptors. If an identified residue was not
conserved in ORF74 it was introduced as a point mutation. Finally, serial truncation
21
constructs of the N and C terminus of the receptor were generated based on homology
with other chemokine and viral receptors.
Table 2 Mutagenesis summary
Description Number
Improved
Expression
Point mutations
Mimic residue in stabilized receptor 18 S93G, L258V
Mimic conserved residue in crystallized class a
receptors 3 L169W
Mimic conserved residue in CXCR 7 -
Remove glycosylation sites 5 -
Stabilize transition between TM7 and helix 8 6 L320E
Deletio
ns
ΔN5, 10, 15, 25, 30, 35 6 -
ΔC2, 5, 8, 13 4 ΔC2, 5
In total 44 unique variations were tested by transient transfection in 293 cells and
comparing expression to WT receptor by western blot. Transfections were repeated at
least three times and mutations consistently displaying greater than twofold increase in
expression over WT as
measured by Western blot
relative signal intensity were
selected as hits. A
representative experiment is
shown in Figure 3. This approach led to the identification of four unique mutations
which increased expression levels of receptor during transient transfection.
48
35
Flag-ORF74
Figure 3 Western blot of transfected 293T cells
expressing ORF74 point mutants
22
Screening fusion partners
for protein stability
Next, I sought to identify soluble
proteins which could be fused to
the third intracellular loop of
ORF74 to promote protein
stability as well as combined
with identified point mutations.
Initially three different fusion
partners which have all been
successfully used to
crystallize GPCRs were
tested in combination with the
previously identified point
mutations. I found that a BRIL
fusion led to the greatest
increase in protein expression
and that this fusion could be
combined with previously identified point mutations to further increase expression
(Figure 4). To validate the ability of these mutations to increase the quality of purified
receptor, I transfected 4x10cm plates of 293T with various ORF74 constructs, purified
the receptor and analyzed the quality and quantity of protein by size exclusion
chromatography (SEC). In accordance with the expression data, introducing a BRIL
48
35
63
Flag-ORF74
Figure 4 Western blot of transfected 293T cells
expressing ORF74 fusion partners
0
0.2
0.4
0.6
0.8
1
1.2
5 7 9
A280 mAu
Time (min)
ORF74– WT
ORF74– S93G, L258V
ORF74– BRIL
ORF74– BRIL, S93G, L258V
Figure 5 SEC to assess protein yield and
homogeneity
23
fusion in combination with S93G and L258V point mutations increased the yield and
monodispersity of purified protein (Figure 5). Further mutational screens were
performed to test fusion junction sites as well as an N-terminal fusion partner. Analysis
of purified mutants by SEC revealed that an ICL3 BRIL fusion to be the most
homogenous construct and that a triple mutation combined with BRIL dramitically
ICL3-BRIL
N-BRIL
N-T4L
Figure 6 Assess fusion optimization and mutagenesis using purified protein
0
20
40
60
80
100
2 3 4 5
Normalized A280
Time (m)
ICL3 vs N fusion
0
20
40
60
80
100
2 3 4 5
Normalized A280
Time (m)
Junction Walking
R-BRIL-TKLQAR
RT-BRIL-KLQAR
RTK-BRIL-LQAR
RTKL-BRIL-QAR
RTKLQ-BRIL-AR
RTKLQA-BRIL-R
ICL3-BRIL
N-BRIL
N-T4L
250
150
100
75
50
37
25
0
5
10
15
20
25
30
5 7 9
A280
Time (min)
Mutation Combination
L258V-BRIL
L170-L258V-L320E-BRIL
L258V-BRIL
L170-L258V-
L320E-BRIL
24
increased yield of purified receptor as assessed by commasie staining and SEC (Figure
6).
Systematic mutagenesis to generate covalent ligand complex
There are no known small molecule ligands that bind to ORF74 and attempts to
stabilize ORF74 with molecules that bind other chemokine receptors were unsuccessful
as assessed by an increase of yield, mono dispersity and thermalstability. Further
attempts to copurify ORF74 with known Chemokine ligands also failed. Table 3
provides a summary of the results of these experiments.
Table 3 Initial screen for stabilizing ligands
Ligand Class Ligand
Name
When Added Copurified Increase
Yield
Increase
Homogeneity
Increase
Tm
Small Molecule AMG-487 During solubilization n/a No No No
Small Molecule MSX-122 During solubilization n/a No No No
Small Molecule SCH 527123 During solubilization n/a No No No
Small Molecule SRT 3109 During solubilization n/a No No No
Small Molecule Reparixin During solubilization n/a No No No
Chemokine vMIP-II During solubilization
& Coexpressed
No No No No
Chemokine CXCL1 Coexpressed No No No No
The inability to copurify a stable complex between ORF74 and a diffusible ligand led me
to pursue methods to generate a covalent complex between ORF74 and known ligand
25
CXCL1. The extracellular environment is an
oxidizing environment, as a result when two free
sulfhydryl groups contained in cysteine residues
are in close proximity, they can spontaneously
form a disulfide bond. Thus, introducing a pair of
cysteine mutations in two separate proteins which
interact in an oxidizing environment can lead to the generation of a covalent complex
(Figure 7). This principle has previously been used to generate a complex between
CXCR4 and vMIP-II that was able to crystallize (Qin, et al., 2015). In the case of CXCR4
the structure of the receptor bound to a small molecule had been solved prior to that of
the ligand complex, thus existing structural data as well as extensive mutagenesis
studies of the receptor were used to develop rationale pairs of cysteine residues.
However, for ORF74 very few specific residues have been empirically shown to be
required for chemokine binding. In fact, only a single manuscript at this time exists
showing a point mutation that abrogates the tyrosine sulfation of the receptor N
terminus and causes of a loss of CXCL1 binding (Feng, Sun, Farzan, & Feng, 2010).
As this is the only existing data regarding a specific ligand interaction, we chose use
CXCL1 as a target for disulfide trapping. Further, this manuscript demonstrated the
functional importance of this complex by showing that ORF74 which could not bind
CXCL1 lost its oncogenic potential. To design cysteine mutants, I relied on the
canonical model for chemokine binding to receptors in which there are two primary
interaction surfaces. It is hypothesized that the receptor N terminus acts as an arm to
first grab the N-loop and 40s-loop of the chemokine. Once the receptor has a hold of the
ORF74 CXCL1
SH
H
SH
ORF74-
CXCL1
complex
Figure 7 Concept of covalent
receptor/ligand complex
26
chemokine, the chemokine N terminus enters the receptor ligand pocket and interacts
with the receptor transmembrane region and transmembrane extracellular loops. Known
receptor ligand binding hotspots where selected for systematic cysteine mutation
(Figure 8). After introducing cysteine mutations ligand and receptor were coexpressed
pair-wise by transient transfection in 293T cells and evaluated for disulfide formation. In
total 106 unique pairs were screened. The molecular weight of CXCL1 is approximately
11 kDa and the molecular weight of ORF74bril is approximately 55 kDa. Thus a
covalent complex run a non-reducing SDS-PAGE gel should produce a band of 66 kDa
when immunoblotted for either CXCL1 or ORF74. Treatment of such a sample with a
A S V A T
E
L
R
C
Q
C
L
Q
5
10
T
L
15
Q
G
I
H
P
20
K
N
I
Q
S
25
V
N
V
K
S
30
P
G
P
H
C
A 35
Q
T
E
V
I
T
40
L
K
45
G
R
K
A
C
50
L
N P
A
P
55
S
A
N
I
K
V
Beta
Sheet
30s
Loop
40s
Loop
N-Term
N Loop
ORF74
N-Term
ORF74
ECL2
I I
K E
K M
L
N
K
D
S
S
N
S
F
N
G
S
Y
D
Y
G
S
C
V
S
V
E
L
E
M
40
30
N
E
Y
C
M
A
K
Q
A
A
T
M
N
G
A
D
W
200
45 pairs
7 pairs
12 pairs
16 pairs
Residues mutated
to cysteine
106 total pairs
CXCL1
R
R
T
V
L
H
V
ORF74
TM5
7 pairs
8 pairs
3 pairs
ORF74
TM6
D
8 pairs
Figure 8 Residues mutated to cysteine in ORF74 and CXCL1, and combinations
chosen for screening.
27
reducing agent should completely eliminate the presence of this band. Finally, in
accordance with my previous data, pull down of receptor should also pull-down
chemokine only when an intact disulfide bond is present. I used these principles to
validate the formation of a disulfide complex.
The initial screen identified 12 putative hits which are summarized in Figure 9 and were
selected for further characterization. The results from this screen reveal important
details about the nature of the interaction between ORF74 and CXCL1. They establish a
critical role for the receptor N terminus, especially the region adjacent to the tyrosine
sulfation sites, for binding to the receptor. The extensive interactions between the N
terminus are of interest from a structural perspective because none of the solved
chemokine/chemokine receptor complexes contain information in this region (Figure
HA-CXCL1
Flag-ORF74
75
50
37
75
50
37
75
50
37
HA-CXCL1
Flag-ORF74
75
50
37
ORF74 WT
D27
D27
D27
Y28
S29
G30
N31
F32
F32
V36
Y197
Y197
CXCL1
WT
G51
P54
N56
G51
N56
N56
N56
I52
N56
A84
H68
A70
HIS PD WCL
K
A S V A T
E
L
R
C
Q
C
L
Q
5
10
T
L
15
Q
G
I
H
P
20
N
I
Q
S
25
V
N
V
K
S
30
P
G
P
H
C
A 35
Q
T
E
V
I
T
40
L
K
45
G
R
K
A
C
50
L
N P
A
P
55
S
A
N
I
K V
Beta
Sheet
30s
Loop
40s
Loop
N-Term
N Loop
ORF74
N-Term
ORF74
ECL2
I I
K E
K M
L
N
K
D
S
S
N
S
F
N
G
S
Y
D
Y
G
S
C
V
S
V
E
L
E
M
40
30
N
E
Y
C
M
A
K
Q
A
A
T
M
N
G
A
D
W
200
Residues mutated
to cysteine
CXCL1
Figure 9 Summary of top disulfide forming cysteine pairs as assessed by Western blot
of whole cell lysate and histidine pull down of receptor. Glycosylation of receptor impairs
clear detection of band shift upon disulfide formation.
28
10). Unlike for other receptors where truncation of the N terminus has minimal effect on
ligand binding, truncation of even 5 amino acids from ORF74 caused a reduction in
disulfide complex formation, and truncation of 10 residues resulted in a dramatic loss of
complex formation for all tested cysteine pairs. Collectively the data established the
ability of the receptor to bind to the chemokine using to distinct modules that are in
accordance with canonical receptor chemokine binding and establish a critical role for
the receptor N terminus.
Figure 10 Structural and sequence alignment of chemokine receptor complexes and
ORF74 chemokine ligands (PDB: 4RWS, 5UIW, 4XT1).
One of the unique features of ORF74 is its ability to bind promiscuously to a broad
range of chemokine ligands. However, the mechanisms underlying this binding are
poorly understood. I hypothesized that I could use disulfide trapping to determine if their
vMIP-II
CXCR4-
T4L
CCL5
CCR5-
rubiredo
xin
CX3CL1
US28
Homologous to CXCL1-N56C disulfide trap residue
CXCL1 ELRCQCLQTLQ-GIHPKNIQSVNVKSPGPHCAQTEVIATLK-NGRKAC
CXCL2 ELRCQCLQTLQ-GIHLKNIQSVKVKSPGPHCAQTEVIATLK-NGQKAC
CXCL3 ELRCQCLQTLQ-GIHLKNIQSVNVRSPGPHCAQTEVIATLK-NGKKAC
CXCL4 DLQCLCVKTTS-QVRPRHITSLEVIKAGPHCPTAQLIATLK-NGRKIC
CXCL5 ELRCVCLQTTQ-GVHPKMISNLQVFAIGPQCSKVEVVASLK-NGKEIC
CXCL6 ELRCTCLRVTL-RVNPKTIGKLQVFPAGPQCSKVEVVASLK-NGKQVC
CXCL7 ELRCMCIKTTS-GIHPKNIQSLEVIGKGTHCNQVEVIATLK-DGRKIC
CXCL8 ELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLS-DGRELC
CXL10 TVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRC
CXCL12 SYRCPCRFFES-HVARANVKHLKIL-NTPNCALQIVARLKN-NNRQVC
Agonist
Neutral Agonist
Inverse Agonist
Disulfide residue is last
position before beta sheet 1 in
ORF74 chemokine ligands
CXCL1-
N56
A
B
29
existed a conserved binding region that is involved in the interaction between ORF74
and all its known chemokine ligands. To examine this, I first performed a sequence and
structural alignment of the known ORF74 ligands (Figure 10b) and found that one of
the top hits for disulfide complex formation (CXCL1-N56C to ORF74-G30C) is the last
residue before a highly conserved beta sheet in all ORF74 ligands. Next, I cloned the
chemokine ligands with a cysteine mutation at this position and systematically
coexpressed them with 7 sequential cysteine mutations in ORF74 N terminus for 56
unique combinations (Figure 11).
Figure 11 Diagram of 56 unique cysteine pair ligand screen and summary of results
with CXCL7 shown as an example.
I found that all chemokines regardless of their function in regulating ORF74 activity
(agonist, neutral, inverse agonist) could form a complex with the receptor in the three
amino acid region Y28, S29, G30. Shown as an example is CXCL7 which showed
highest efficiency in this region but was also able to complex with additional residues as
well.
C
C
C
C
Beta
Sheet
30s
Loop
40s
Loop
N-Term
N Loop
ORF74
N-Term
Screen 56 Disulfide Pairs
F
N
G
S
Y
D
Y
7 pairs per
chemokine
CXCL 1/2/3/5/7/8/10/12
30
CXCL7-N76C
ORF74:
Y26C
D27C
Y28C
S29C
G30C
N31C
F32C
150
100
75
50
37
25
250
Flag-ORF74
HA-CXCL7
150
100
75
50
37
25
250
HIS Pull down
30
Purification of covalent receptor-ligand complex
I next sought to purify receptor-ligand complexes for biochemical characterization and
validation. To do so I conducted small scale purifications by costransfecting receptor
and ligand constructs in 4x10cm dishes of 293 cells and purified the receptor from the
150
100
75
50
37
25
20
15
10
150
100
75
50
37
25
20
15
10
ORF74-3M-G30C
+ + +
CXCL1-N56C
- + +
10-Histidine Rec Rec Lig
DTT
- + - + - +
Flag-ORF74
HA-CXCL1
ORF74-CXCL1
CXCL1-HA-10His
CXCL1-HA
ORF74-CXCL1
ORF74
30
80
130
180
230
20 40 60 80
Fluorescence a.u.
Temperature (°C
CPM
A2A
ORF74
ORF74-CXCL1
ORF74
ORF74-G30C
CXCL1-N56C
- +
Coomassie
250
150
100
75
50
37
0
2
4
6
8
10
12
14
16
5.5 6.5 7.5 8.5 9.5
A280
Time (min)
SEC
ORF74
ORF74-CXCL1
A B
C D
HIS Pull down
Figure 12 Purification, validation, and biochemical characterization of covalent
ORF74-CXCL1 complex. A) Pull down of either receptor or ligand using histidine
tag to monitor band shift in the presence of reducing conditions B) Coomassie stain
of protein purified apo and complexed with ligand. C) Evaluation of protein by SEC.
D) Evaluation of thermal denaturing of apo and complexed receptor using CPM dye
31
membrane fraction of these cells. I specifically aimed to validate that the complex I
purified could be disrupted by addition of a reducing agent to the purified protein prior to
running on an SDS-PAGE gel. I conducted three separate purifications, receptor alone,
receptor pull down with chemokine coexpression, and chemokine pull down with
receptor coexpression (Figure 12a). The results demonstrate that complex formation is
specific, highly efficient and is sensitive to reduction by DTT. Next, I assessed the
impact of complex formation on the biochemical characteristics of the receptor by
measuring protein purity, yield, homogeneity, and thermalstability. Coexpression of
CXCL1 and ORF74 containing the appropriate cysteine pair resulted in a greater than 2-
fold increase in protein yield as assessed by both Coomassie staining and SEC (Figure
0
20
40
60
80
100
5 6 7 8 9 10
Normalized mAu
Time (min)
0
2
4
6
8
10
12
5 6 7 8 9 10
mAu
Time (min)
ORF74-G30C
CXCL1-N56C
CXCL2-N56C
CXCL3-N56C
CXCL7-N76C
CXCL12-N43C
250
150
100
75
50
37
75
50
37
+PNGase F
ORF74-CXCL
Complex
ORF74
0
20
40
60
80
100
5 6 7 8 9 10
Normalized mAu
Time (min)
0
2
4
6
8
10
12
5 6 7 8 9 10
mAu
Time (min)
CXCL1
CXCL3
CXCL7
CXCL1
CXCL3
CXCL7
75
50
37
ORF74-
G30C
CXCL1
CXCL2
CXCL3
CXCL7
75
50
37
HA-CXCL
Flag-ORF74
ORF74-
G30C
CXCL1
CXCL2
CXCL3
CXCL7
A B
Figure 13 Purification of ORF74 in complex with different chemokine ligands. A)
Complex efficiency and purity evaluated after deglycosylation of receptor with PNGase.
B) SEC used to evaluate complex monodispersity
32
12a-c). However, the receptor complex was less homogenous, as it had two peaks in
contrast to a single peak for apo receptor when evaluated by SEC. Despite that, when
protein quality was assessed by CPM assay to determine thermalstability, only receptor
37°C DDM
30°C DDM
Coomassie
0
20
40
60
80
100
4 6 8 10 12
Normalized A280
Time (minutes)
SEC
37°C DDM
30°C DDM
0
20
40
60
80
100
20 40 60 80
Normalized Intensity (A.U.)
Temperature (°C)
Thermal Stability
0
20
40
60
80
100
5 7 9
Normalized A280
Time (min)
SEC
DDM
LMNG
DDM
LMNG
A B
C D
E
Figure 14 Optimization of expression and membrane extraction of ORF74-CXCL1
complex. A) Comparison of protein purified from same number of cells cultured at
different temperatures. B) Evaluation of protein homogeneity. C) Protein solubilized and
purified in different detergents. D) Evaluation of stability of samples from C. E) Chemical
structure of two detergents tested.
33
ligand complex exhibited a melting curve transition with a sinusoidal slope similar to the
thermalstable A2A receptor (Figure 12d). In contrast APO receptor had a nearly linear
increase in signal as temperature increased suggesting that the receptor did not
uniformly unfold and is likely more unstable than the complex. I next sought to purify
and characterize additional ligand complexes (Figure 13a). The yield for ORF74-
CXCL1 was consistently higher than for other complexes, however the monodispersity
for a complex of ORF74-CXCL7 was superior. These initial small-scale purifications
provided rationale for developing a system to express the receptor in suspension culture
in order to generate sufficient amounts of protein to pursue crystallization trials.
Optimizing expression in mammalian cells
The first step I took to optimize expression was to check receptor expression when 293
cells are cultured at 30°C after transfection. Culture of mammalian cells at reduced
temperature can improve protein yield and the percentage of protein that is properly
folded (Al-Fageeh, Marchant, Carden, & Smales, 2006). At lower temperatures cell
growth is dramatically reduced and the expression of chaperone proteins is increased. I
conducted a time course of receptor expression when cells were cultured at 37°C or
moved to 30°C 16h post transfection and evaluated receptor expression by western
blot. I found that receptor expression was consistently higher in cells cultured at 30°C
and that the optimal time for harvest was 72 hours post transfection. Through small
scale purifications I determined a 2-3 fold increase of ORF74-CXCL1 yield when cells
were cultured at 30°C (Figure 14a). This protein also had greater homogeneity (Figure
14b).
34
Next, I hypothesized that due to ORF74 having high basal activity
and being in complex with an agonist, its stability might be increased
when extracted from membranes with lauryl maltose neopentyl
glycol detergent (LMNG) (Figure 14e). This idea came from the
observation that other agonist bound receptors are more stable in
this detergent as it has a slower off rate than the detergent I was
currently using dodecyl maltoside (DDM) (Chae, et al., 2011).
Extraction and purification of the ORF74-CXCL1 complex from
membranes in either LMNG or DDM was compared. Both methods
yielded comparable amounts of protein, however the monodispersity
and thermalstability of receptor was improved in LMNG micelles
(Figure 14c-d).
In order to efficiently scale up protein expression, the 293S GnTI(-) cell line was chosen
for the fact that it is already suspension adapted as well as for its lack of the enzyme
(GnTI) required for formation of complex glycosylation (Reeves, Callewaert, Contreras,
& Khorana, 2002). My previous analysis of purified receptor and treatment with
deglycosylation enzyme PNGase revealed that ORF74 is heterogeneously glycosylated
(Figure 13a). Downstream crystallization requires homogeneity and glycans can
interfere with crystal formation. To address this I attempted to remove glycosylation
sites by mutagenesis, but as seen elsewhere, this severely attenuated protein
expression (Wu, et al., 2015). Additionally, PNGase treatment of purified receptor
decreased protein homogeneity as assessed by SEC. Comparison of ORF74
Coomassie
250
150
100
75
50
37
293S GnTI(-)
293T
Figure 15
Comparison of
ORF74-CXCL1
purified from
different cells
35
expression in 293T and 293S GnTI(-) cells by immunoblot revealed comparable
expression levels, and disulfide complex formation efficiency was similar. However
where expression of receptor in 293T cells revealed the presence of multiple bands
corresponding to different glycosylation states, receptor expressed in 293S GnTI(-) cells
migrated as a single band by SDS-PAGE analysis (Figure 15). Thus, 293S cells were
grown in suspension culture on an orbital platform, receptor and ligand were
cotransfected, and protein was purified from the membrane fraction.
Receptor purified from suspension was pure as assessed by Coomassie l;staining and
was mostly monodisperse (Figure 16a). However, while the portion of receptor in a
disulfide complex with CXCL1 was greater 90% in small scale purification, it was less
0
20
40
60
80
100
20 40 60 80
Normalized Fluorescence a.u.
Temperature (°C
Thermalstability
0
20
40
60
80
100
120
0 2 4 6 8 10 12
A280 mAu
Time (min)
SEC
75
50
37
25
20
100
150
250
CXCL1-HA Flag-ORF74
75
50
37
25
20
100
150
250
Coomassie
75
50
37
25
20
100
150
250
ORF74-CXCL1
ORF74
C
C
Flag
ORF74-G30C
C
CXCL1-N56C
C
Flag
ORF74-G30C
10HIS
10HIS
HIS Purification
• Receptor
• Receptor Complex
Flag Purification:
• Receptor Complex
Receptor/Complex
Complex only
CXCL1-HA
CXCL1-Flag
Flag-ORF74-10His +
ORF74-10His +
ORF74-CXCL1
ORF74
ORF74-CXCL1
Coomassie
Flag
A
B
C
D
CXCL1-N56C
Figure 16 Strategy to isolate receptor ligand complex from apo receptor A) Analysis of
receptor based purification by SDS-PAGE and SEC B) Comparison of principle for ligand
versus receptor based purification C) SDS-PAGE validation of ligand based purification D)
Comparison of protein stability between to purification methods
36
than 50% when purified from large
scale culture (compare Figure 13a to
Figure 16a). This necessitated the
development of a method to specifically
isolate receptor-ligand complex. I
chose to put a purification tag on the
ligand so that the ligand could be
directly pulled down from the
membrane fraction of cells (Figure
16b). In theory, high salt washing of
this membrane fraction should remove
any ligand not covalently bound to the receptor. Thus, when the ligand is purified only
covalent ligand receptor complex will be isolated. Any apo receptor will not be captured.
As a proof of principle Flag affinity purification was used to purify CXCL1 from the
membrane fraction of cells also expressing ORF74 and compared to the previous
purification protocol using histidine purification pulling down receptor. Figure 16c shows
that by pulling down the ligand a pure population of receptor chemokine complex can be
isolated. Further, this complex has greater thermalstability than a sample which
contains a mix of apo and ligand bound receptor (Figure 16d). While this method was
successful in generating pure complex it reduced the yield of protein to 100ug/L, too low
to pursue crystallization trials. To increase protein expression of 293S cells baculovirus
mediated transduction (BacMam) was used (Figure 17) (Goehring, et al., 2014).
CMV
promoter
ORF74
pEZT
1. Clone
2. Transform into E. coli
BmDH10Bac
3. Isolate Bacmid
4. Transfect Sf9
5. Amplify/titer
virus
6. Infect 293S
Figure 17 Strategy for BacMam
transduction of mammalian cells relies on
the principle that baculovirus infects but
doesn’t replicate in mammalian cells and
that a mammalian specific CMV promoter
can drive transgene expression in infected
cells.
37
I first generated BacMam viruses containing ORF74 fused to YFP at the C terminus so
that I could easily monitor protein expression and optimize infection conditions. Virus
was produced and titered in Sf9 cells. Titered virus was used to infect 293S GnTI(-)
cells in suspension culture at various multiplicities of infection (MOI) in the presence or
absence of the histone deacetylase inhibitor valproic acid (VPA). Expression was
evaluated by flow cytometry to determine the condition that produced both the highest
infectivity and median fluorescent intensity for ORF74-YFP (Figure 18a). The optimal
condition of MOI 5 in the presence of 2.2mM was compared to transfection of
suspension cells with plasmid DNA containing ORF74-YFP using two different
transfection protocols. The expression of ORF74-YFP was significantly higher by
baculovirus transduction than by transfection (Figure 18b). These results were further
validated by expressing ORF74 and CXCL1 in cells by transfection or by baculovirus
and purifying the complex. The protein yield was higher for BacMam transduction as
shown in Figure 18c. A large-scale purification using this system was performed and
the protein yield was greater than 400ug/L and of sufficient homogeneity to be used for
crystallization trials (Figure 19).
38
0.1 1 10 100
0
5000
10000
MOI
Median Fluorescent Intensity
ORF74-YFP
Mock
2.2mM VPA
5mM VPA
0.1 1 10 100
0
50
100
MOI
% Positive
ORF74-YFP
Mock
2.2mM VPA
5mM VPA
Transfect: ORF74-YFP
Mock
Transfection 1
+2.2mM VPA
Transfection 2
+2.2mM VPA
72h post transfection
Infect: ORF74-YFP
Uninfected
MOI 5
MOI 5 +
2.2mM VPA
MOI 5 +
5mM VPA
72h post transduction
Transfect
BacMam
2
nd
IMAC + +
Coomassie
A
B C
Figure 18 Evaluation of BacMam mediated ORF74 expression. A) Flow cytometry
analysis of cells infected with baculovirus carrying ORF74-YFP under control of a CMV
promoter. Cells were infected at various MOIs and treated with valproic acid (VPA). B)
Comparison of ORF74-YFP expression in cells when expressed in suspension cells by
transient transfection or by baculovirus transduction. C) ORF74 and CXCL1 were
coexpressed by indicated method and purified with purification tag on the ligand. Protein
was then run on an 15% gel and analyzed by Coomassie blue staining. Complex is
indicated by arrow.
39
Figure 19 Example of purification from optimized expression conditions using
baculovirus transduction of suspension 293S cells as well as the optimal receptor and
ligand constructs from extensive screening.
Expression of ligand-receptor complex in insect cells
Extension optimization of receptor expression led to the ability to purify homogenous
and stable ORF74-CXCL1 complex in mammalian cells, however expression and
purification from mammalian cells is costly due to the requirement of specialized
suspension culture media and FBS. Further yield in mammalian cells is limited by the
fact although baculovirus can infect these cells it cannot replicate. In contrast, insect
cells can be grown in relatively more cost-effective protein and serum free media. Since
they facilitate baculovirus replication, protein expression in theory can also be higher.
Thus, I cloned both ORF74 and CXCL1 into a pFastBac plasmid carrying the
baculovirus polyhedron promoter for expressing and purifying the complex from Sf9
cells. To validate the quality of protein purified from insect cells I purified the exact same
receptor and ligand constructs in parallel from mammalian and insect cells (Table 7:
#207, #261. Table 8: #216, #260). Protein was purified using the same methodology and
0
20
40
60
80
100
2 3 4 5
Normalized A280
Time (min)
C
CXCL1-N56C
C
10HIS
ORF74bril-G30C-L170W-
L258V-L320E
ORF74-CXCL1
DTT - +
Coomassie
ORF74
CXCL1
BRIL
40
quality of protein was characterized by SEC, SDS-PAGE, and thermalstability assay.
The two proteins had nearly indistinguishable SEC profiles, thermalstability, and band
pattern on SDS-PAGE (Figure 20a-d). The only significant difference between the two
samples was that the yield and purity of protein was higher for the insect cell purification
(Table 4: 20190327). Next, I took advantage of the higher yield and purity of the insect
cell expression platform to more clearly investigate the combinatorial thermalstabilizing
role of individual point mutations and different ICL3 fusion partners to develop an
optimized thermal stable receptor complex (Figure 21a-d).
Figure 20 Purification of protein from insect Sf9 and mammalian 293S GnTI(-) cells A)
Monodispersity analysis by SEC of two protein samples with BSA as a reference. B)
SDS-PAGE followed by coomasie blue staining to compare molecular weight and purity
C) Thermalstability measured by CPM assay with previously used bril fusion as a
reference. D) First derivative of the curve generated in C was used to quantify melting
temperature (Tm) of the two samples.
20 40 60 80
0
50
100
Normalize of First derivative of CPM
XYL Tm = 59.8°C
BRIL Tm = 54.8°C
293S XYL Tm = 59.8°C
0
20
40
60
80
100
0 5 10 15
Normalized mAu
Time (min)
SEC
BSA
Sf9 ORF74xyl-CXCL1
293S ORF74xyl-CXCL1
20 40 60 80
0
200
400
600
800
Temperature (°C)
Fluorescence (a.u.)
Tm Assay
XYL
BRIL
293S XYL
Sf9 293s
DTT - + - +
250
150
100
75
50
37
25
20
15
10
*ORF74xyl-CXCL1
**ORF74xyl
***CXCL1
*
**
***
Sf9 ORF74xyl-CXCL1
293S ORF74xyl-CXCL1
293S ORF74bril-CXCL1
Sf9 ORF74xyl-CXCL1
293S ORF74xyl-CXCL1
BSA
Sf9 Tm = 59.8°C
293S Tm = 59.8°C
A
B
C
D
41
Crystallization trials
The most successful strategy for crystallizing membrane proteins has been to use the
lipid cubic phase (LCP), where purified protein is reconstituted in a synthetic lipid bilayer
and overlaid with a precipitant solution (Figure 22) (Caffrey, A comprehensive review of
the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins
and complexes, 2015). A major caveat with this approach is that diffusion of the protein
in LCP, unlike in an aqueous environment, cannot be assumed. As a result, fluorescent
recovery after photobleaching (FRAP) has been used to verify the diffusion, a
necessary requirement for crystallization, of a target protein in LCP. FRAP requires that
0
20
40
60
80
100
20 40 60 80
Normalized Fluorescence
Temperature (C)
Combination
G30C
G30C-L169W
G30C-L169W-L258V-XYL
0
20
40
60
80
100
20 40 60 80
Normalized Fluorescence
Temperature (C)
ICL3 Fusions
PGS
RUBR
T4L
XYL
No ICL3
0
20
40
60
80
100
20 40 60 80
Normalized Fluorescence
Temperature (C)
Point Mutations
G30C
G30C-L169W
G30C-L258V
G30C-L320E
A
B
C
D
Figure 21 Thermalstability assay with CPM dye using ORF74-CXCL1 complexes purified
from insect cell 40ml cultures. A) No ICL3 fusion and individual point mutations. B) No
point mutations and different fusion partners. C) Combination of point mutations and
fusion partners. D) Snake plot of optimal CXCL1 (grey circles) and ORF74 (white circles)
constructs (Table 7 #216, and #260 respectively).
42
the target protein is fluorescently labeled and for GPCRs the
conventional approach has been to label the receptor with an
amine reactive dye at pH 7.2 to preferentially label the N
terminus (Fenalti, Abola, Wang, Wu, & Cherzov, 2015). The
logic is that N terminal labeling should have minimal effect on
the protein structure, solubility or diffusion and relies on the
fact that most GPCRs are purified in the presence of diffusible
small molecules which do not contain an amine group. In
contrast, the ORF74-CXCL1 complex contains two separate N
terminal amine groups, one on the receptor and one on the
ligand. For all three solved chemokine/chemokine receptor
structures, the N terminus of the chemokine is buried in the
receptor ligand binding pocket (PDB: 4RWS, 5UIW, 4XT1).
During repeated labeling experiments with the amine reactive
cyanine 3 NHS ester, aggregation of the ORF74-CXCL1
Express
ORF74/CXCL1
in cell membrane
Purify
ORF74/
CXCL1
in detergent
micelle
Crystallize
ORF74/CXCL1 in lipid cubic phase
Figure 22 Processing of receptor complex for crystallization (LCP image
reproduced from (Caffrey, Crystallizing membrane proteins for structure–
function studies using lipidic mesophases, 2011)
Cy3
ORF74xyl-
CXCL1
OR74-
CXCL1
DTT - + - +
* **
***
* **
***
Coomassie Cy3 Figure 23 Two
ORF74-CXCL1
samples were
purified and one was
labeled with Cy3.
Image is samples on
SDS-PAGE +/-
reducing conditions
and imaged with
either bright field or
fluorescence. (*
ORF74-CXCL1
**ORF74 ***CXCL1)
43
complex was observed by SEC analysis. Further, fluorescence imaging of the complex
run under reducing conditions revealed labeling of CXCL1 as well as likely labeling of
copurifying lipids carrying amine groups at the dye front (Figure 23). As a control
unlabeled protein was run on the same gel and fluorescence signal was only observed
in samples labeled with Cy3. As a result, receptor diffusion in LCP could not be
conclusively determined by labeling.
Figure 24 Method for setting up crystallization trials and validating purified complex
using ORF74-G30C-L169W-L258V-Flag + CXCL1-N56C-3C-10his purified from Sf9
cells as a template.
44
Biomasses from a culture volume greater than 0.5L were
used for purifying protein to be used for crystallization trials.
In total 24 separate protein expressions at this scale in
either mammalian or insect cells were conducted (described
in Table 4). The general workflow is provided in Figure 24a
and the methods used to evaluate purity, quantity,
homogeneity, and stability of the purified protein are shown in Figure 24b-d. After
reconstitution in LCP, protein was overlaid with precipitant solution and monitored for
crystal formation by bright field and cross polarized light. Crystals have been observed
to grow in a single condition (Figure 25) from sample shown in Figure 24. However, it
has not yet been determined if these are protein or salt. Efforts to validate and
reproduce these crystals are currently ongoing.
Figure 25 image of
crystals from sample
shown in Figure 24
45
Table 4 Summary of Large-Scale Purifications
Date
Receptor Ligand
Pull
down
Enzymatic
Treatment
Volume
(L) Cells
Expressed
by Detergent
Total
ug ug/L
mg/ml
final ID ICL3 Mutation Tags N/C Gene ID Mutation Tag C
20171101 130 BRIL L169W-L258V-L320E Flag/3C-His CXCL1 C15 N56C HA R 3C/EndoH 0.8 293T Transfection DDM/CHS 38 47.5 3.8
20171117 130 BRIL L169W-L258V-L320E Flag/3C-His CXCL1 C15 N56C HA R 3C/EndoH 1 293S GnTI(-) Transfection DDM/CHS 60 60 6
20171130 130 BRIL L169W-L258V-L320E Flag/3C-His CXCL12 CX12 N43C HA R 3C/EndoH 1 293S GnTI(-) Transfection DDM/CHS 96.4 96.4 9.64
10171214 130 BRIL L169W-L258V-L320E Flag/3C-His CXCL12 CX12 N43C HA R - 1 293S GnTI(-) Transfection DDM/CHS n.d. n.d. n.d.
20180131 130 BRIL L169W-L258V-L320E Flag/3C-His CXCL1 C15 N56C HA R 3C 2 293S GnTI(-) Transfection DDM/CHS 155.6 77.8 15.56
20180131 130 BRIL L169W-L258V-L320E Flag/3C-His CXCL12 CX12 N43C HA R 3C 2.4 293S GnTI(-) Transfection DDM/CHS 66 27.5 3
20180222 130 BRIL L169W-L258V-L320E Flag/3C-His CXCL7 CX7 N76C HA R 3C 2.4 293S GnTI(-) Transfection MNG/CHS 124.4 51.8 12.44
20180416 187 BRIL L169W-L258V-L320E-dC3 -/- CXCL1 187 N56C Flag L - 2 293S GnTI(-) Transfection MNG/CHS 187 93.5 8.5
20180612 191 BRIL L169W-L258V-L320E-dC3 -/- CXCL1 191 N56C 3C-His L 3C/EndoH 5.9 293S GnTI(-) Transfection MNG/CHS 264 44.7 12
20180718 191 BRIL L169W-L258V-L320E-dC3 -/- CXCL1 191 N56C 3C-His L 3C 3.6 293S GnTI(-) Transfection MNG/CHS 146 40.6 5.85
20180718 205 BRIL L169W-L258V-L320E-dC3 -/- CXCL1 207 N56C 3C-His L 3C 3 293S GnTI(-) BacMam MNG/CHS 673 224 26.9
20180803 205 BRIL L169W-L258V-L320E-dC3 -/- CXCL1 207 N56C 3C-His L 3C 2 293S GnTI(-) BacMam MNG/CHS 901 451 43
20180803 205 BRIL L169W-L258V-L320E-dC3 -/- CXCL1 207 N56C 3C-His L 3C/EndoH 2 293S GnTI(-) BacMam MNG/CHS 854 427 41
20180816 212 - L169W-L258V-L320E-dC3 -/- CXCL1 207 N56C 3C-His L 3C/EndoH 2.4 293S GnTI(-) BacMam MNG/CHS 546 227.5 26
20180907 212 - L169W-L258V-L320E-dC3 -/- CXCL1 207 N56C 3C-His L - 1.6 293S GnTI(-) BacMam MNG/CHS 411 257 16
20180926 205 BRIL L169W-L258V-L320E-dC3 -/- CXCL12 214 N43C 3C-His L
3 293S GnTI(-) BacMam MNG/CHS 88 29 4
20181017 205 BRIL L169W-L258V-L320E-dC3 -/- CXCL1 207 N56C 3C-His L 3C/PNGase 3.6 293S GnTI(-) BacMam MNG/CHS 584 162 26.54
20181101 215 BRIL L169W-L258V-L320E-dC3 -/- CXCL1 216 N56C 3C-His L 3C/PNGase 1 Sf9 Baculovirus MNG/CHS 491 491 22.3
20190306 251 XYL L169W-L258V-dC2 -/Flag CXCL1 216 N56C 3C-His L 3C 4 Sf9 Baculovirus MNG/CHS 300 75 10
20190314 251 XYL L169W-L258V-dC2 -/Flag CXCL1 216 N56C 3C-His L 3C 3 Sf9 Baculovirus MNG/CHS 315 105 13.11
20190327 251 XYL L169W-L258V-dC2 -/Flag CXCL1 216 N56C 3C-His L 3C 4 Sf9 Baculovirus MNG/CHS 630 158 21
20190327 261 XYL L169W-L258V-dC2 -/Flag CXCL1 207 N56C 3C-His L 3C 4 293S GnTI(-) BacMam MNG/CHS 420 105 14
20190425 248 PGS L169W-L258V-dC2 -/Flag CXCL1 216 N56C 3C-His L 3C 4 Sf9 Baculovirus MNG/CHS 1150 289 25.55
46
Description of Expression Constructs
Table 5 Constructs for transient transfection of receptor in mammalian cells
ID Vector Tag Fusion Location Linker ORF74 Mutation JUNCTION 1ST ORF 2ND ORF
13 pIRES N-Flag/C-10His - - - G88S - ORF74 -
14 pIRES N-Flag/C-10His - - - S93G - ORF74 -
15 pIRES N-Flag/C-10His - - - V104D - ORF74 -
16 pIRES N-Flag/C-10His - - - M106D - ORF74 -
17 pIRES N-Flag/C-10His - - - Y128S - ORF74 -
18 pIRES N-Flag/C-10His - - - Y130S - ORF74 -
19 pIRES N-Flag/C-10His - - - S135C - ORF74 -
20 pIRES N-Flag/C-10His - - - S140C - ORF74 -
21 pIRES N-Flag/C-10His - - - L258V - ORF74 -
22 pIRES N-Flag/C-10His - - - T299I - ORF74 -
23 pIRES N-Flag/C-10His - - - S307I - ORF74 -
24 pIRES N-Flag/C-10His - - - Y326A - ORF74 -
25 pIRES N-Flag/C-10His - - - WT - ORF74 -
26 pIRES N-Flag/C-10His - - - DRY - ORF74 -
27 pIRES N-Flag/C-10His - - - NpxxY - ORF74 -
28 pIRES N-Flag/C-10His - - - S93G-L258V - ORF74 -
29 pIRES N-Flag/C-10His BRIL ICL3 SA-SA - RRTK-BRIL-LQARR ORF74 -
30 pIRES N-Flag/C-10His T4L ICL3 SA-SA - RRTK-T4L-LQARR ORF74 -
31 pIRES N-Flag/C-10His RUBR ICL3 SA-SA - RRTK-RUBR-LQARR ORF74 -
32 pIRES N-Flag/C-10His T4L ICL3 SA-SA S93G RRTK-T4L-LQARR ORF74 -
33 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G RRTK-BRIL-LQARR ORF74 -
34 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L258V RRTK-BRIL-LQARR ORF74 -
35 pIRES N-Flag/C-10His RUBR ICL3 SA-SA S93G RRTK-RUBR-LQARR ORF74 -
36 pIRES N-Flag/C-10His RUBR ICL3 SA-SA L258V RRTK-RUBR-LQARR ORF74 -
37 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V RRTK-BRIL-LQARR ORF74 -
38 pIRES N-Flag/C-10His T4L ICL3 SA-SA S93G-L258V RRTK-T4L-LQARR ORF74 -
39 pIRES N-Flag/C-10His RUBR ICL3 SA-SA S93G-L258V RRTK-RUBR-LQARR ORF74 -
40 pIRES N-Flag/C-10His - - - S307T - ORF74 -
41 pIRES N-Flag/C-10His - - - Y326F - ORF74 -
42 pIRES N-Flag/C-10His - - - F134W - ORF74 -
43 pIRES N-Flag/C-10His - - - A172N - ORF74 -
44 pIRES N-Flag/C-10His - - - V249D - ORF74 -
45 pIRES N-Flag/C-10His - - - V252P - ORF74
46 pIRES N-Flag/C-10His - - - S93G-L258V-F134W - ORF74 -
47 pIRES N-Flag/C-10His - - - S93G-L258V-V249D - ORF74 -
48 pIRES N-Flag/C-10His - - - S93G-L258V-L252P - ORF74 -
47
49 pIRES N-Flag/C-10His - - - S93G-L258V-V249D-V252P - ORF74 -
50 pIRES N-Flag/C-10His - - - S93G-L258V-F134W-V249D - ORF74 -
51 pIRES N-Flag/C-10His - - - S93G-L258V-F134W-V252P - ORF74 -
52 pIRES N-Flag/C-10His - - - S93G-L258V-F134W-V249D-V252P - ORF74 -
53 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA S93G-L258V-F134W RRTK-BRIL-LQARR ORF74 -
54 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA S93G-L258V-V249D RRTK-BRIL-LQARR ORF74 -
55 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA S93G-L258V-L252P RRTK-BRIL-LQARR ORF74 -
56 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA S93G-L258V-V249D-V252P RRTK-BRIL-LQARR ORF74 -
57 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA S93G-L258V-F134W-V249D RRTK-BRIL-LQARR ORF74 -
58 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA S93G-L258V-F134W-V252P RRTK-BRIL-LQARR ORF74 -
59 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA S93G-L258V-F134W-V249D-V252P RRTK-BRIL-LQARR ORF74 -
28-1 pIRES N-Flag/C-10His-AviTag - - - S93G-L258V
ORF74 -
28-2 pIRES N-10His-Flag/C-AviTag - - - S93G-L258V
ORF74 -
37-1 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA S93G-L258V RRTK-BRIL-LQARR ORF74 -
37-2 pIRES N-10His-Flag/C-AviTag BRIL ICL3 SA-SA S93G-L258V RRTK-BRIL-LQARR ORF74 -
60 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA F134W RRTK-BRIL-LQARR ORF74 -
61 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA V249D RRTK-BRIL-LQARR ORF74 -
62 pIRES N-Flag/C-10His-AviTag BRIL ICL3 SA-SA V252P RRTK-BRIL-LQARR ORF74 -
63 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V RR-BRIL-TKLQARR ORF74 -
64 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V RRT-BRIL-KLQARR ORF74 -
65 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V RRTKL-BRIL-QARR ORF74 -
66 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V RRTKLQ-BRIL-ARR ORF74 -
67 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V RRTKLQA-BRIL-RR ORF74 -
68 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN22 RRTK-BRIL-LQARR ORF74 -
69 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L91D RRTK-BRIL-LQARR ORF74 -
70 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L94D RRTK-BRIL-LQARR ORF74 -
71 pIRES N-Flag/C-10His BRIL N SA-SA - DDGA-BRIL-PAAE ORF74 -
72 pIRES N-Flag/C-10His T4L N SA-SA - DDGA-T4L-PAAE ORF74 -
73 pIRES N-Flag/C-10His RUBR N SA-SA - DDGA-RUBR-PAAE ORF74 -
74 pIRES N-Flag-GFP/C-10His BRIL ICL3 SA-SA S93G-L258V DDGA-GFP-PAAE ORF74 -
75 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-K192C RRTK-BRIL-LQARR ORF74 -
76 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-Q193C RRTK-BRIL-LQARR ORF74 -
77 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-A194C RRTK-BRIL-LQARR ORF74 -
78 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-M195C RRTK-BRIL-LQARR ORF74 -
79 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-Y197C RRTK-BRIL-LQARR ORF74 -
80 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-E198C RRTK-BRIL-LQARR ORF74 -
81 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-N199C RRTK-BRIL-LQARR ORF74 -
82 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-A200C RRTK-BRIL-LQARR ORF74 -
83 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93C-L258V RRTK-BRIL-LQARR ORF74 -
84 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-A79C RRTK-BRIL-LQARR ORF74 -
48
85 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-Q323C RRTK-BRIL-LQARR ORF74 -
86 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-Q323A-R324A-M325A RRTK-BRIL-LQARR ORF74 -
87 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L54Y RRTK-BRIL-LQARR ORF74 -
88 pIRES N-Flag/C-10His BRIL ICL3 SA-SA I82T RRTK-BRIL-LQARR ORF74 -
89 pIRES N-Flag/C-10His BRIL ICL3 SA-SA N92A RRTK-BRIL-LQARR ORF74 -
90 pIRES N-Flag/C-10His BRIL ICL3 SA-SA N111W RRTK-BRIL-LQARR ORF74 -
91 pIRES N-Flag/C-10His BRIL ICL3 SA-SA D178P RRTK-BRIL-LQARR ORF74 -
92 pIRES N-Flag/C-10His BRIL ICL3 SA-SA A308C RRTK-BRIL-LQARR ORF74 -
93 pIRES N-Flag/C-10His BRIL ICL3 SA-SA C316F RRTK-BRIL-LQARR ORF74 -
94 pIRES C-Flag-10His BRIL ICL3 SA-SA L258V RRTK-BRIL-LQARR ORF74 -
95 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-G25C RRTK-BRIL-LQARR ORF74 -
96 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-Y26C RRTK-BRIL-LQARR ORF74 -
97 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-D27C RRTK-BRIL-LQARR ORF74 -
98 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-Y28C RRTK-BRIL-LQARR ORF74 -
99 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-S29C RRTK-BRIL-LQARR ORF74 -
100 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-G30C RRTK-BRIL-LQARR ORF74 -
101 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-N31C RRTK-BRIL-LQARR ORF74 -
102 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-F32C RRTK-BRIL-LQARR ORF74 -
103 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93D RRTK-BRIL-LQARR ORF74 -
104 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L169W RRTK-BRIL-LQARR ORF74 -
105 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC5-L258V RRTK-BRIL-LQARR ORF74 -
106 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V RRTK-BRIL-LQARR ORF74 -
107 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC13-L258V RRTK-BRIL-LQARR ORF74 -
108 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN10 RRTK-BRIL-LQARR ORF74 -
109 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L320E RRTK-BRIL-LQARR ORF74 -
110 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-V36C RRTK-BRIL-LQARR ORF74 -
111 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-S37C RRTK-BRIL-LQARR ORF74 -
112 pIRES N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V-V38C RRTK-BRIL-LQARR ORF74 -
113 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L258V-E35C RRTK-BRIL-LQARR ORF74 -
114 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L258V-L169W RRTK-BRIL-LQARR ORF74 -
115 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L258V-L320E RRTK-BRIL-LQARR ORF74 -
116 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V-L169W RRTK-BRIL-LQARR ORF74 -
117 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V-L320E RRTK-BRIL-LQARR ORF74 -
118 pIRES N-Flag BRIL ICL3 SA-SA S93G-L258V-Y197C RRTK-BRIL-LQARR ORF74 -
119 pIRES N-Flag BRIL ICL3 SA-SA S93G-L258V-V36C RRTK-BRIL-LQARR ORF74 -
120 pIRES N-Flag BRIL ICL3 SA-SA S93G-L258V-G30C RRTK-BRIL-LQARR ORF74 -
121 pIRES N-Flag BRIL ICL3 SA-SA S93G-L258V-F32C RRTK-BRIL-LQARR ORF74 -
122 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
123 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
124 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V-Y28C RRTK-BRIL-LQARR ORF74 -
49
125 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V-S29C RRTK-BRIL-LQARR ORF74 -
126 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V-G30C RRTK-BRIL-LQARR ORF74 -
127 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V-F32C RRTK-BRIL-LQARR ORF74 -
128 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-L258V-V36C RRTK-BRIL-LQARR ORF74 -
129 pIRES N-CXCL1/C-10His BRIL ICL3 SA-SA ΔC8-L258V RRTK-BRIL-LQARR ORF74 -
130 pIRES N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
131 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
132 pIRES N-Flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
133 pIRES N-Flag BRIL ICL3 SA-SA ΔC8-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
134 pIRES N-Flag/C-10His BRIL ICL3 SA-SA V36C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
135 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔC8-V36C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
136 pIRES N-Flag BRIL ICL3 SA-SA V36C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
137 pIRES N-Flag BRIL ICL3 SA-SA ΔC8-V36C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
138 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN5-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
139 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN10-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
140 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN15-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
141 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN21-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
142 pIRES N-CXCL1/C-10His BRIL ICL3 SA-SA L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
143 pIRES N-CXCL1/C-10His BRIL ICL3 SA-SA L258V-L169W-L320E-ΔC8 RRTK-BRIL-LQARR ORF74 -
144 pIRES N-CXCL1/C-10His BRIL ICL3 SA-SA L258V-L169W-L320E-ΔC5 RRTK-BRIL-LQARR ORF74 -
145 pIRES N-Flag/C-10His BRIL ICL3 SA-SA L258V-L169W-L320E-ΔC5 RRTK-BRIL-LQARR ORF74 -
146 pIRES N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC5 RRTK-BRIL-LQARR ORF74 -
147 pIRES N-Flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC5 RRTK-BRIL-LQARR ORF74 -
148 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN10-L258V-L169W-L320E-ΔC5 RRTK-BRIL-LQARR ORF74 -
149 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN10-G30C-L258V-L169W-L320E-ΔC5 RRTK-BRIL-LQARR ORF74 -
150 pIRES N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E (NoSP) RRTK-BRIL-LQARR ORF74 -
151 pIRES N-Flag/C-10His BRIL ICL3 SA-SA G30C-L169W-L258V RRTK-BRIL-LQARR ORF74 -
152 pIRES N-Flag/C-10His BRIL ICL3 SA-SA G30C-S93G-L169W-L258V RRTK-BRIL-LQARR ORF74 -
153 pIRES N-Flag/C-10His BRIL ICL3 SA-SA G30C-S93G-L170W-L258V RRTK-BRIL-LQARR ORF74 -
154 pIRES N-Flag/C-10His BRIL ICL3 SA-SA G30C-S93G-L169W-L170W-L258V RRTK-BRIL-LQARR ORF74 -
155 pIRES N-Flag/C-10His BRIL ICL3 SA-SA R208C-L169W-L258V RRTK-BRIL-LQARR ORF74 -
156 pIRES N-Flag/C-10His BRIL ICL3 SA-SA R212C-L169W-L258V RRTK-BRIL-LQARR ORF74 -
157 pIRES N-Flag/C-10His BRIL ICL3 SA-SA D274C-L169W-L258V RRTK-BRIL-LQARR ORF74 -
158 pIRES C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
159 pIRES N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR ORF74 -
160 pIRES N-Flag/C-10His BRIL ICL3 SA-SA R208C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
161 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN5-R028C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
162 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN10-R028C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
163 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN15-R028C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
164 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN20-R028C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
50
165 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN25-R028C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
166 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN30-R028C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
167 pIRES N-Flag/C-10His BRIL ICL3 SA-SA ΔN35-R028C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
168 pVitro2 N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
169 pVitro2 N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 CXCL1-N56C-HA
170 pVitro2 Ligand-C-flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL2-N56C ORF74
171 pVitro2 Ligand-C-flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL3-N56C ORF74
172 pVitro2 Ligand-C-flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL5-M62C ORF74
173 pVitro2 Ligand-C-flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL7-N76C ORF74
174 pVitro2 Ligand-C-flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL8-F48C ORF74
175 pVitro2 Ligand-C-flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL10-S44C ORF74
176 pVitro2 Ligand-C-flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL12-N43C ORF74
177 pIRES N-Flag/C-10His BRIL ICL3 SA-SA N18Q-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
178 pIRES N-Flag/C-10His BRIL ICL3 SA-SA N22Q-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
179 pIRES N-Flag/C-10His BRIL ICL3 SA-SA N31Q-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
180 pIRES N-Flag/C-10His BRIL ICL3 SA-SA N202Q-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
181 pIRES N-Flag/C-10His BRIL ICL3 SA-SA N18,22Q-G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
182 pVitro2 N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR ORF74 CXCL1-N56C-HA
183 pVitro2 N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 ORF74
184 pVitro2 N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL1-N56C-HA
185 pVitro2 Ligand-C-HA BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL1-N56C ORF74
186 pIRES - BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR ORF74 -
187 pVitro2 - BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL1-N56C-Flag ORF74
188 pVitro2 - BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL4-C-Flag ORF74
189 pVitro2 - BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL6-C-Flag ORF74
190 pVitro2 - BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR vMIP-II-L47C-Flag ORF74
191 pVitro2 - BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL1-N56C-3C-10His ORF74
192 pVitro2 - - - - G30C-L258V-L169W-L320E - CXCL1-N56C-3C-10His ORF74
193 pVitro2 N-Flag - - - G30C-L258V-L169W-L320E - CXCL1-N56C-3C-10His ORF74
194 pVitro2 N-Flag/C-8His - - - G30C-L258V-L169W-L320E-ΔC3 - CXCL1-N56C-HA ORF74
195 pVitro2 N-Flag/C-10His - - - L258V-L169W-L320E-ΔC3 - CXCL1-N56C-HA ORF74
196 pIRES - - - - G30C-L258V-L169W-L320E-ΔC3 - ORF74 -
197 pIRES N-Flag - - - G30C-L258V-L169W-L320E-ΔC3 - ORF74 -
198 pIRES N-Flag/C-10His - - - G30C-L258V-L169W-L320E-ΔC3 - ORF74 -
199 pVitro2 N-Flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL1-N56C-3C-10His ORF74
200 pVitro2 N-Flag/C-10His BRIL ICL3 SA-SA L258V-L169W-L320E-ΔC3 RRTK-BRIL-LQARR CXCL1-N56C-HA ORF74
201 pVitro2 C-3c-YFP BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR CXCL1-N56C-3C-10His ORF74
202 pVitro2 C-3c-mCherry BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR CXCL1-N56C-3C-10His ORF74
203 pIRES N-Flag/C-3c-mCherry BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
204 pIRES N-Flag/C-3c-mCherry BRIL ICL3 SA-SA ΔN35-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74 -
51
Table 6 Constructs for transient transfection of ligands in mammalian cells
ID Vector Tag Location Mutation 1ST ORF 2ND ORF
V1 pIRES HA C - vMIP-II -
V2 pIRES HA C G25C vMIP-II -
V3 pIRES HA C A26C vMIP-II -
V4 pIRES HA C S27C vMIP-II -
V5 pIRES HA C W28C vMIP-II -
V6 pIRES HA C H29C vMIP-II -
V7 pIRES HA C R30C vMIP-II -
V8 pIRES HA C P31C vMIP-II -
V1b pCDNA HA C - vMIP-II -
V2b pCDNA HA C G25C vMIP-II -
V3b pCDNA HA C A26C vMIP-II -
V4b pCDNA HA C S27C vMIP-II -
V5b pCDNA HA C W28C vMIP-II -
V6b pCDNA HA C H29C vMIP-II -
V7b pCDNA HA C R30C vMIP-II -
V9 pIRES 3C-Human Fc C - vMIP-II -
V10 pIRES 3C-Human Fc C QQEE vMIP-II -
C1 pIRES HA - - CXCL1 -
C2 pIRES HA - S36C CXCL1 -
C3 pIRES HA - V37C CXCL1 -
C4 pIRES HA - A38C CXCL1 -
C5 pIRES HA - T39C CXCL1 -
C6 pIRES HA - E40C CXCL1 -
C7 pIRES HA - L41C CXCL1 -
C8 pIRES HA - R42C CXCL1 -
C9 pIRES HA - Q50C CXCL1 -
C10 pIRES HA - G51C CXCL1 -
C11 pIRES HA - I52C CXCL1 -
C12 pIRES HA - H53C CXCL1 -
C13 pIRES HA - P54C CXCL1 -
C14 pIRES HA - K55C CXCL1 -
C15 pIRES HA - N56C CXCL1 -
C16 pIRES HA - G81C CXCL1 -
C17 pIRES HA - R82C CXCL1 -
C18 pIRES HA - K83C CXCL1 -
C19 pIRES HA - A84C CXCL1 -
C20 pIRES HA - H68C CXCL1 -
C21 pIRES HA - A70C CXCL1 -
C22 pIRES 3C-Human Fc - - CXCL1 -
C23 pIRES HA - Q44C CXCL1 -
C24 pIRES HA - L46C CXCL1 -
C25 pIRES HA - Q47C CXCL1 -
C26 pIRES HA - T48C CXCL1 -
C27 pIRES HA - L49C CXCL1 -
C28 pIRES HA - Q71C CXCL1 -
C29 pIRES HA - L86C CXCL1 -
C30 pIRES HA-10HIS - N56C CXCL1 -
C31 pIRES HA-10HIS - A70C CXCL1 -
C32 pIRES HA-10HIS - A84C CXCL1 -
C33 pIRES HA-10HIS - G51C CXCL1 -
C34 pIRES HA-10HIS - Q47C CXCL1 -
C35 pIRES HA-10HIS - L49C CXCL1 -
C36 pIRES HA - I57C CXCL1 -
C37 pIRES HA - Q58C CXCL1 -
C38 pIRES Flag - T39C CXCL1 -
C39 pIRES Flag - N56C CXCL1 -
C40 pIRES 3C-3xFlag - - CXCL1 -
C41 pIRES 3C-3xFlag - T39C CXCL1 -
C41 pIRES 3C-3xFlag - N56C CXCL1 -
C42 pIRES HA - K23C CXCL12 -
C43 pIRES HA - P23C CXCL12 -
C44 pIRES HA - V24C CXCL12 -
C45 pIRES HA - S25C CXCL12 -
C46 pIRES HA - L26C CXCL12 -
52
C47 pIRES HA - S27C CXCL12 -
C48 pIRES HA - Y28C CXCL12 -
C49 pIRES HA - R29C CXCL12 -
C50 pVITRO2 HA/HA - N56C CXCL1 CXCL1
C51 pVITRO2 HA - N56C CXCL1 -
C52 pIRES Flag - N43C CXCL12 -
C53 pIRES 3C-3xFlag - N43C CXCL12 -
C54 pIRES 3C-HA-10His - N56C CXCL1 -
C55 pIRES 3C-10His - N56C CXCL1 -
53
Table 7 Constructs for baculovirus mediated expression of receptor and ligand in
mammalian cells
ID Vector Tag Fusion Location Linker Mutation JUNCTION 1ST ORF
205 pEZT - BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-dC3 RRTK-BRIL-LQARR ORF74
206 pEZT C-3c-YFP BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-dC3 RRTK-BRIL-LQARR ORF74
207 pEZT 3C-10His - - - N56C - CXCL1
208 pEZT N-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-dC3 RRTK-BRIL-LQARR ORF74
209 pEZT - BRIL ICL3 SA-SA G30C-L258V-L169W RRTK-BRIL-LQARR ORF74
210 pEZT C-Flag BRIL ICL3 SA-SA G30C-L258V-L169W-L320E RRTK-BRIL-LQARR ORF74
211 pEZT C-Flag BRIL ICL3 SA-SA G30C-L258V-L169W RRTK-BRIL-LQARR ORF74
212 pEZT C-Flag - - - G30C-L258V-L169W - ORF74
213 pEZT - - - - N76C - CXCL7
214 pEZT 3C-10His - - - N43C - CXCL12
217 pEZT 3C-10His - - - N56C-dC5 - CXCL1
218 pEZT 3C-10His - - - N56C - CXCL2
219 pEZT 3C-10His - - - N56C - CXCL3
220 pEZT 3C-10His - - - H54C - CXCL4
221 pEZT 3C-10His - - - M62C - CXCL5
222 pEZT 3C-10His - - - T62C - CXCL6
223 pEZT 3C-10His - - - F48C - CXCL8
224 pEZT 3C-10His - - - S44C - CXCL10
225 pEZT 3C-10His - - - L47C - vMIP-II
226 pEZT - RUBR ICL3 SA-SA - RRTK-RUBR-LQARR ORF74
227 pEZT - T4L ICL3 SA-SA - RRTK-T4L-LQARR ORF74
228 pEZT - BRIL ICL3 SA-SA G30C RRTK-BRIL-LQARR ORF74
229 pEZT - - - - G30C - ORF74
261 pEZT C-Flag XYL ICL3 SA-SA G30C-L258V-L169W-ΔC2 RRTK-XYL-LQARR ORF74
54
Table 8 Constructs for baculovirus mediated expression of receptor and ligand in
insect cells
ID Vector Tag Fusion Location Linker Mutation JUNCTION 1ST ORF
1 pFastBac1 N-Flag/C-10His - - - -
ORF74
2 pFastBac1 N-Flag/C-10His BRIL ICL3 - - RRTK-BRIL-LQARR ORF74
3 pFastBac1 N-Flag/C-10His BRIL ICL3 -SA - RRTK-BRIL-LQARR ORF74
4 pFastBac1 N-Flag/C-10His BRIL ICL3 SA-SA - RRTK-BRIL-LQARR ORF74
5 pFastBac1 N-Flag/C-10His T4L ICL3 -SA - RRTK-T4L-LQARR ORF74
6 pFastBac1 N-Flag/C-10His T4L ICL3 SA-SA - RRTK-T4L-LQARR ORF74
7 pFastBac1 N-Flag/C-10His T4L ICL3 - - RRTK-T4L-LQARR ORF74
8 pFastBac1 N-Flag/C-10His RUBR ICL3 -SA - RRTK-RUBR-LQARR ORF74
9 pFastBac1 N-Flag/C-10His RUBR ICL3 SA-SA - RRTK-RUBR-LQARR ORF74
10 pFastBac1 N-Flag/C-10His RUBR ICL3 - - RRTK-RUBR-LQARR ORF74
11 pFastBac1 N-Flag/C-10His - - - DRY
ORF74
12 pFastBac1 N-Flag/C-10His - - - NPxxY
ORF74
28 pFastBac1 N-Flag/C-10His - - - S93G-L258V
ORF74
33 pFastBac1 N-Flag/C-10His BRIL ICL3 SA-SA S93G RRTK-BRIL-LQARR ORF74
34 pFastBac1 N-Flag/C-10His BRIL ICL3 SA-SA L258V RRTK-BRIL-LQARR ORF74
37 pFastBac1 N-Flag/C-10His BRIL ICL3 SA-SA S93G-L258V RRTK-BRIL-LQARR ORF74
38 pFastBac1 N-Flag/C-10His T4L ICL3 SA-SA S93G-L258V RRTK-T4L-LQARR ORF74
39 pFastBac1 N-Flag/C-10His RUBR ICL3 SA-SA S93G-L258V RRTK-RUBR-LQARR ORF74
215 pFastBac1 - BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-dC3 RRTK-BRIL-LQARR ORF74
216 pFastBac1 3C-10His - - - N56C - CXCL1
230 pFastBac1 C-Flag - - - G30C-L258V-L169W - ORF74
231 pFastBac1 C-Flag - - - G30C - ORF74
232 pFastBac1 C-Flag - - - G30C-L169W - ORF74
233 pFastBac1 C-Flag - - - G30C-L258V - ORF74
234 pFastBac1 C-Flag - - - G30C-L320E - ORF74
235 pFastBac1 C-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-L320E-dC3 RRTK-BRIL-LQARR ORF74
236 pFastBac1 3C-10His - -
N56C-dC5 - CXCL1
237 pFastBac1 3C-10His - -
N56C - CXCL2
238 pFastBac1 3C-10His - -
N56C - CXCL3
239 pFastBac1 3C-10His - -
H54C - CXCL4
240 pFastBac1 3C-10His - -
M62C - CXCL5
241 pFastBac1 3C-10His - -
T62C - CXCL6
242 pFastBac1 3C-10His - -
N76C - CXCL7
243 pFastBac1 3C-10His - -
F48C - CXCL8
244 pFastBac1 3C-10His - -
S44C - CXCL10
245 pFastBac1 3C-10His - -
N43C - CXCL12
246 pFastBac1 3C-10His - -
L47C - vMIP-II
247 pFastBac1 C-Flag BRIL ICL3 SA-SA G30C-L258V-L169W-ΔC2 RRTK-BRIL-LQARR ORF74
248 pFastBac1 C-Flag PGS ICL3 SA-SA G30C-L258V-L169W-ΔC2 RRTK-PGS-LQARR ORF74
249 pFastBac1 C-Flag RUBR ICL3 SA-SA G30C-L258V-L169W-ΔC2 RRTK-RUBR-LQARR ORF74
250 pFastBac1 C-Flag T4L ICL3 SA-SA G30C-L258V-L169W-ΔC2 RRTK-T4L-LQARR ORF74
251 pFastBac1 C-Flag XYL ICL3 SA-SA G30C-L258V-L169W-ΔC2 RRTK-XYL-LQARR ORF74
252 pFastBac1 C-Flag/C-10His BRIL ICL3 SA-SA G30C-L258V-L169W-ΔC2 RRTK-BRIL-LQARR ORF74
253 pFastBac1 C-Flag/C-10His BRIL ICL3 SA-SA L258V-L169W-ΔC2 RRTK-BRIL-LQARR ORF74
254 pFastBac1 C-Flag/C-10His - - - L258V-L169W-ΔC2
ORF74
255 pFastBac1 C-Flag/C-10His - - - -
ORF74
256 pFastBac1 - PGS ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC2 RRTK-PGS-LQARR ORF74
257 pFastBac1 - XYL ICL3 SA-SA G30C-L258V-L169W-L320E-ΔC2 RRTK-XYL-LQARR ORF74
258 pFastBac1 - XYL ICL3 SA-SA G30C-L169W-ΔC2 RRTK-XYL-LQARR ORF74
259 pFastBac1 - XYL ICL3 SA-SA G30C-L169W-L258V-ΔC2 RRTK-XYL-LQARR ORF74
260 pFastBac1 - XYL ICL3 SA-SA G30C-L169W-L320E-ΔC2 RRTK-XYL-LQARR ORF74
55
Chapter 3 Identification of nanobodies that bind ORF74
56
Introduction
GPCRs are challenging targets for antibody development due to the fact that they have
minimal antigenic surface exposed for binding to soluble proteins as most of the protein
is embedded in the membrane. Further, it is difficult to purify sufficient quantity and
quality of membrane proteins for immunization. As a result, there are no high quality
commercially available antibodies which recognize ORF74. Antibodies against cell
surface proteins are useful for both diagnostic and therapeutic purposes, and an
antibody recognizing ORF74 would be valuable to the KSHV research field as well as a
potential therapeutic agent for patients with KS.
Yeast nanobody surface display
The thermalstablization of ORF74 and optimization of
expression in mammalian cells ultimately allowed for the
purification of sufficient amounts of apo receptor to make
antibody discovery possible. I specifically chose to use
purified ORF74 to identify a camelid heavy chain only
antibody which could bind the receptor. This type of
antibody is called a “nanobody” and has multiple
advantages over conventional heavy and light chain
containing immunoglobulins. The unique extended nature
of the complimentary determining regions (CDRs) of
nanobodies can reach into buried hydrophobic pockets,
such as the ligand binding pocket of a GPCR, more
efficiently than conventional antibodies. Further,
CDR3
CDR1
CDR2
Stalk
Yeast Cell
Figure 26 Architecture of a
nanobody displayed on the
surface of a yeast cell,
colored complimentary
determining regions (CDRs)
determine antigen
specificity (McMahon, et al.,
2018).
57
nanobodies tend to recognize specific structural conformations, such as those exhibited
by multipass transmembrane proteins, rather than linear epitopes. In addition to these
features, because nanobodies are encoded by a single polypeptide they can be
functionally expressed in bacteria and yeast. This characteristic allows the screening of
nanobodies which bind an antigen using library display technologies (Figure 26). The
ability to perform in vitro screening of nanobodies bypasses the need for animal
immunization, which reduces the time and costs for identifying novel antibodies. I used
purified ORF74 to take advantage of a recently developed synthetic nanobody library
displayed on yeast (McMahon, et al., 2018) to screen and identify ORF74 specific
nanobodies.
Selection Strategy
The general principle of
yeast selection is
illustrated in Figure 27. A
library with 1x10^8 unique
structurally designed
nanobody clones is
expressed on the surface
of yeast cells. Cells are
mixed with purified antigen
to allow binding to
individual nanobody
Repeat
Bind to antigen Naïve library
Isolate bound antigens
(FACS/MACS)
Amplify Figure 27 Strategy for isolating antigen specific nanobody
clones expressed on the surface of yeast
58
clones. Yeast bound by the antigen are then labeled with a fluorophore or magnetic
bead for specific isolation and amplification. This process is repeated until over 20% of
the yeast cells bind to the target antigen. My goal was to obtain a nanobody which
specifically recognized the extracellular portion of ORF74 as this would have potential
use as a therapeutic or diagnostic tool. The specific workflow I used relied on negative
selection against ORF74 covalently bound to CXCL1 (Figure 28). Negative selection
with ORF74-CXCL1 was used to deplete nanobodies which could bind to the BRIL
fusion on the receptor ICL3 in addition to regions outside of the receptor ligand pocket.
Three rounds of MACS followed by two rounds of FACS were performed and 55
individual clones were isolated and sequenced.
Figure 28 Selection strategy for isolating nanobodies which bind to the extracellular
ligand binding region of ORF74
Library
Flag-Antibody
ORF74 Apo (Flag)
ORF74 Apo (FITC)
Round 3
Flag-
ORF74-CXCL1
ORF74 Apo (Cy5)
Round 4
ORF74 Apo
Round 2
Round 1
Round 5
ORF74 Apo
FACS
SORT
Screen
Clones
FITC-Flag-
ORF74-CXCL1
ORF74
BRIL
ORF74
BRIL
CXCL1
ORF74
BRIL
Nb
59
Summary of ORF74 nanobody screening
Figure 29 A) Sequence alignment of CDR regions of nanobodies recognizing ORF74.
B) Flow cytometry analysis of purified receptor binding to an individual yeast clone. C)
Schematic of protein complexes used for validating yeast clones
Individual yeast clones were first validated by testing their ability to specifically bind apo
receptor and not ORF7-CXCL1 complex or the M2-Flag antibody used for labeling
during selection. Of the 55 screened clones, 7 unique nanobody sequences were
identified. Of the unique sequences 4 showed specific binding and represented 89% of
all hits. Of those 4 unique clones 3 had multiple hits. The 3 specific clones which
Antibody Only
CXCL1-ORF74bril
ORF74bril
CXCL1-ORF74bril
ORF74bril
Gate Nby +
Gate Nb +
ORF74-Flag-FITC
CDR1 CDR2 CDR3
Nb1 (17hits) GNIST-YYM EFVAGISLGASTNY AAGGYDLSWYSY
Nb2 (6hits) GSISVGIYM EFVAGISYGGTTNY AAGLYQASSYGY
Nb3 (25hits) GNISSDVYM EFVAGISYGSTTNY AVGYGHGSGYDY
* * * * * * * * *
3/13 Highly Variable Residues
6/10 Partially Variable Residues
9/23 Total conserved
Flag
Y
⍺FlagFITC
Flag
Y
⍺FlagFITC
Yeast
Nb
ORF74
BRIL
CXCL1
ORF74
BRIL
A
B C
60
showed biding only to ORF74 apo were selected for purification and biochemical
characterization (Figure 29).
Biochemical characterization of nanobodies
Nanobodies 1-3 were expressed and purified from bacterial cells and protein was
labeled for using to check binding to receptor expressed on the surface of mammalian
cells to further validate binding (Figure 30a). Lysine labeling was chosen due to the
presence of four lysine residues on each nanobody located exclusively outside of the
antigen binding region. Labeled nanobody was used to determine on cell EC50 values
Periplasm
Column f.t.
Wash 1
Wash 2
Elution
250
150
100
75
50
37
25
20
15
10
General Nanobody Structure
CDR
Lysines
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0
20
40
60
80
100
Log Antibody (nM)
% Positive Cells
% Positive Comparison
Nb1-ORF74-3M-Bril
Nb1-Mock
Flag-ORF74-3M-Bril
Flag-Mock
293 Cell
ORF74
Nb
SPR
KD (nM)
Nanobody 1 311
Nanobody 2 96
Nanobody 3 237
Tm
°C
ORF74 52.17
ORF74 + Nb 3 55.03
B A C
D E F
Figure 30 Summary of nanobody purification and binding to ORF74. A) Protein
purified from e. coli periplasm. B) Location of lysine residues on nanobody surface
in relation to antigen binding CDR region. C) Strategy for validating binding of
purified nanobody to ORF74 by flow cytometry. D) Flow cytometry analysis of cells
expressing either ORF74 or mock and stained with nanobody of Flag antibody.
ORF74 contained an N terminal flag tag as a control. E) In vitro affinity was
measured using a Nicoya openSPR. F) Measurement of thermalstability of apo
ORF74 and an in vitro formed complex of ORF74 and nanobody 3.
61
to determine affinity for each nanobody to native receptor expressed in a membrane
environment. All three nanobodies had affinity between 20-30nM (Figure 30b-d).
Affinity was also determined between purified nanobody and purified receptor, and the
thermalstability of receptor was higher when bound to a nanobody (Figure 30e). Finally
gel filtration was performed to verify the stable formation of a complex. Nanobody 3
coeluted with ORF74.
Figure 31 Gel filtration fractions run on SDS-PAGE gel of either purified Nanobody 3,
ORF74, or Nanobody 3 and ORF74 copurified together.
Summary
I used a yeast display library to isolate and characterize three unique nanobodies which
are able to bind ORF74 both in vitro and in a native membrane environment.
Elution Volume (mL)
7.2
7.6
8
8.4
8.8
9.2
9.6
10
10.4
10.8
11.2
11.6
12
12.4
Elution Volume (mL)
14.8
15.2
15.6
16
16.4
16.8
17.2
17.6
18
18.4
18.8
19.2
19.6
20
Nanobody 3
ORF74
Nanobody 3
+ ORF74
62
Chapter 4 Conclusions
63
Summary and future plans for structural and antibody studies
A;fter extensive screening of 344 unique constructs and different expression platforms I
identified conditions for producing pure, thermalstable ORF74 ligand covalent complex
at quantities sufficient for pursuing crystallization trials. This work provides the
foundation for further pursuit of high throughput crystallization screens for identifying
conditions to grow protein crystals that would facilitate determination of an atomic
structure of the ORF74 oncogene. I have identified a single condition where crystals are
growing and am in the process of validating and reproducing this sample. I also plan to
repeat crystallization trials of receptor with ICL3 fusions of BRIL and PGS at higher final
protein concentrations by purifying protein from a larger biomass. Further I will conduct
another round of nanobody screening to identify a nanobody that could bind and
stabilize the ORF74-CXCL1 complex to facilitate crystallization of the receptor in an
active state. In order to further investigate the extracellular nanobodies I identified, I will
perform cell signaling assays with ORF74 expressing cells. Specifically, I will test the
ability of the nanobodies to either activate or inhibit ORF74 dependent G protein and
arrestin signaling pathways. If any of the nanobodies are able to inhibit signaling they
will be evaluated in an in vivo mouse model to evaluate their potential efficacy as a
therapeutic. Collectively the data provided in chapters two and three provide the first
extensive characterization of an ORF74 specific antibody and provide the foundation for
future structural studies.
64
Chapter 5 Materials and Methods
65
Cells
293S GnTi- cells were purchased from ATCC (CRL-3022). Cells were cultured in
DMEM + 10% FBS + 1% P/S when grown as adherent cells in 10cm dishes in a 37c
incubator with 5% CO2. Suspension cells were cultured FreeStyle Media (Gibco) + 2%
FBS + 1% P/S in baffled polycarbonate flasks cells filled to 1/3 max volume. Flasks
were placed on an orbital shaking platform at 130 RPM in a 37c incubator with 5% CO2.
Cells were regularly counted and split when the density reached 2x10^6 cells/ml to a
final density of 5x10^5 cells/ml.
293T cells were purchased from ATCC (CRL-3216) and were cultured in DMEM + 10%
FBS + 1% P/S when grown as adherent cells in 10cm dishes in a 37c incubator with 5%
CO2.
Sf9 cells were purchased from Expression Systems and cultured in ESF 921 media
without supplementation in polycarbonate flasks (no baffles) in a 27c incubator without
CO2. Cells were regularly counted and split when the density reached 4-8x10^6 cells/ml
to a final density of 7x10^5 cells/ml. Viability was monitored by trypan blues staining to
ensure cells were >90% viable.
DH10bac cells were a gift from the Stevens Lab at the USC Bridge institute. TOP10
cells were purchased from Thermo Fisher (C404010). BL21RIPL cells were a gift from
Byung-Ha OH at KAIST. All E.coli cells were amplified using the rubidium chloride
competent cell protocol.
Transfection
For transfection of adherent 293T or 293S GnTi-, cells were seeded in either 6-well or
10cm dishes to be 80% confluent the following day. On the day of transfection DNA at
1mg/ml was mixed with PEI at 1mg/ml at 1:5 ratio in serum free media, incubated for 15
66
minutes and added dropwise to cells. Efficiency was assessed by performing GFP
transfections and performing flow cytometry.
For transfection of 293S suspension cells two different protocols were used. In the first
protocol cells were pelleted and resuspend in serum free media at a density of 2x10^6
cells/ml. DNA/PEI complexes were prepared as above and added to cells. A second
protocol was later determined to have higher efficiency. Cells were pelleted and
resuspended in serum and antibiotic free RPMI supplemented with 0.1% pluronic at
2x10^7 cells/ml in 50ml final volume. 1.5mg of plasmid DNA and 3mg of PEI were
added sequentially to the cells. Cells were incubated on an orbital shaker for 90 minutes
and then added to 950ml of prewarmed Freestyle media containing 2% FBS + 1% P/S.
Immunoblot
For measuring protein in cell lysates, cells were lysed in a buffer composed of 50mM
HEPES pH7.5, 150mM NaCl, 10% Glycerol, 1% DDM, 0.2% CHS, and protease
inhibitor cocktail. Lysate was mixed with 4xLDS loading dye (no reducing agent) and run
without boiling on 8, 10, 12 or 15% SDS-PAGE gels for 60 minutes at 150v using a
BioRad mini Protean system. Protein was transferred to PVDF membrane (Bio-Rad
Laboratories) by semi-dry transfer at 25V for 30 min. Membranes were blocked in 5%
milk in TBST for 30minutes at room temperature. Primary antibodies Flag (1:2,000;
Sigma-Aldrich), and HA (1:2,000; Covance) were incubated overnight at 4c. After
washing three times for minutes per wash with TBST, HRP-conjugated secondary
antibodies were incubated on membranes at 1:4000 for 45 minutes. Bands were
developed with ECL reagent and imaged on a BioRad Chemidoc imaging system.
67
Recombinant bacmid production
Constructs were cloned into baculovirus donor plasmids pFastBacI and pEZT and
miniprep DNA was isolated using a ThermoFisher GeneJET miniprep kit. DNA
concentration was evaluated by measuring A260 on a Nanodrop. 1ul containing 50ng of
plasmid DNA was added to 35ul of DH10bac cells on ice and incubated for 20 minutes.
After 20 minutes mixture was transferred to a pre-cooled 15ml round bottom tube on
ice. The tube containing DNA/DH10bac mixture was heat shocked at 42c for 45
seconds then placed on ice for 2 minutes. 200uL of S.O.C. media was then added and
mixture was incubated shaking at 37c for 3-4h. After incubation, 3ul of transformation
mixture was mixed with 50ul of LB media and spread on selection plates (LB Agar
containing: 50ug/ml kanamycin, 7ug/ml gentamycin, 10ug/ml tetracyclin, 40ug/ml IPTG,
100ug/ml Bluo-gal). Plates were incubated at 37c for 48h. White colonies were selected
and used to inoculate 3ml cultures of LB (containing 50ug/ml kanamycin, 7ug/ml
gentamycin, 10ug/ml tetracyclin) in 15ml round bottom tubes. Cultures were incubated
at 37c shaking for approximately 16 hours. Cells were collected by centrifugation for 10
minutes at 4000 RPM. Supernatant was discarded, pellet was resuspended in 300ul of
P1 buffer (Qiagen) and transferred to a 1.5ml Eppendorf tube. 300ul of P2 was added,
tubes were gently inverted 4-6 times (never vortexed) and incubated at room
temperature for 5 minutes. 300ul of P3 was added and sample was spun at 14,000 x g
for 10 minutes at 4c. 800ul of supernatant was collected with care taken to not disturb
the pellet, and transferred to a clean Eppendorf tube containing 700ul of isopropanol.
Tubes were inverted to mix, incubated on ice for 10 minutes, and DNA was collected by
centrifugation at 14,000 x g for 15 minutes at 4c. Supernatant was discarded and pellet
68
was resuspended in 500ul of 70% ethanol. Sample was centrifuged at 14,000 x g for 5
minutes at 4c. All liquid was carefully removed from tube using vacuum aspiration and
70ul of milli-q water of water was added to tube. Pipetting was not used for mixing to
avoid damaging bacmid genome and solution was stored at 4c for up to one month.
Production and titering of baculovirus
Adapted from published protocol (Gustavsson, Zheng, & Handel, 2016).
Sf9 cells were split to a density of 7x10^5 cells/ml prior to the day of transfection. On the
day of transfection, they were counted again to ensure a density of 1.2-1.4x10^6. 42ul
of Transfection medium from (Expression Systems 92-020) was mixed with 8ul of
Cellfectin-II (Thermo 10362100). In a separate tube 45ul of Transfection medium was
mixed with 5ul of bacmid DNA. Both reactions were equilibrated at room temperature for
5 minutes, combined and incubated for 30 minutes more at room temperature. 2.5ml of
Sf9 cells at appropriate density were added per well of a 24-well deep well plate that
had been sterilized by autoclaving. 100ul of transfection reaction was added to cells and
plate was covered by an adhesive breathable membrane and incubated at 27c shaking
at 300 RPM for 96 hours. Prior to collecting, a 10ul aliquot of cells was stained with Anti-
GP64-PE and analyzed by flow cytometry to ensure >90% of cells are infected. Plates
were spun at 2000 x g for 15 minutes and the supernatant was transferred to clean 5ml
tubes and labeled P0.
To produce P1 virus cells were split to a density of 1.4x10^6 cells/ml the day prior to
infection. On the day of infection cells were counted to ensure a density of 2-3x10^6
cells/ml. A 125ml flask containing 50ml of cells at appropriate density was infected with
69
500ul of P0 virus and incubated at 27 shaking at 140 RPM for 48 hours. Prior to
collection a small aliquot of cells was used to ensure viability was 50-80% and infectivity
was over 95% as assessed by flow cytometry using Anti-GP64-PE. Cells were
transferred to a 50ml conical and spun at 2000 x g for 15 minutes. Supernatant was
poured into a clean conical, labeled P1, and stored at 4c in the dark.
To titer virus cells were split to a density of 1.4x10^6 cells/ml the day prior to infection.
On the day of infection cells were counted to ensure a density of 2-3x10^6 cells/ml. A
1:250 dilution of virus (4ul P1 virus + 986 ESF 921 media) was prepared in a 24-well
deep well plate. Serial dilutions were prepared of 1:500, 1:1000, and 1:2000 in 500ul
final volume. 2ml of cells were added to each 500ul of virus dilution. The plate was
sealed and incubated at 27c shaking at 300 RPM for 20-24 hours. The percentage of
infected cells was then measured by staining cells with Anti-GP64-PE and analyzing by
flow cytometry. The following equation was used to determine viral titer when less than
30% of cells are positive: Titer = ( (total cell number )* (%GP64/100)* (viral dilution
factor) ) / Volume of viral inoculum. The following equation was used to determine viral
titer when more than 30% of cells are positive: Titer = ( (total cell number )* [-ln(1-
(%GP64/100))] * (viral dilution factor) ) / Volume of viral inoculum. P1 viral titers were
routinely 1x10^9 infectious units/ml.
Flow cytometry
For Anti-GP64 staining 10ul of Sf9 cells at density of 3-5x10^6 cells/ml was mixed with
either 10ul FACS buffer (TBS+10% FBS) containing .04ug of antibody and incubated at
4c for 15minutes. 180ul of FACS buffer was added to cell/antibody mixture and samples
70
were immediately analyzed with the Attune NxT Autosampler. For flag receptor staining
10ul of either Sf9 or 293S cells were mixed with 10ul of 2x Permeablizing Solution 2 (BD
340973) containing .04ug of APC anti-DYKDDDDK (Clone L5 Biolegend 637308) at 4c
for 15 minutes. 180ul of 1x Permeablizing Solution 2 was added to cell/antibody mixture
and samples were immediately analyzed with the Attune NxT Autosampler
Protein expression by baculovirus and purification
For expression in 293S, cells at a density of 2-3x10^6 cells/ml in 2L flasks containing
800ml of cells were coinfected with virus expressing receptor at MOI6 and virus
expressing chemokine at MOI4. After addition of virus cells were incubate at 37°C on
shaking platforms (120-135 RPM) in a CO2 incubater for 8-16 hours. Valproic acid was
then added to cells at a final concentration of 2.2mM and cells were then cultured in a
30°C incubator. 72h post infection cells were collected by centrifugation at 4000 RPM in
500ml conical bottles and cell pellet was stored at -80°C.
For expression in Sf9 cells, cells at a density of 2-3x10^6 cells/ml in 3L flasks containing
1L of cells were coinfected with virus expressing receptor at MOI6 and virus expressing
chemokine at MOI4. 48 hours post infection cells were collected by centrifugation at
4000 RPM in 500ml conical bottles and cell pellet was stored at -80°C.
Cell pellets were suspended in hypotonic buffer (10mM HEPES pH 7.5, 10mM MgCl2,
20mM KCl supplemented with protease inhibitors and benzonase for 293 cells) and
dounced repeatedly. Final volume was raised to 200ml and solution was aliquoted to
Ti70 25ml tubes. Samples were centrifuged in a Ti70 rotor at 42,000 RPM (~180,000 x
g) for 30 minutes. Pellets were resuspended by douncing and pelleted one more time
71
with hypotonic buffer and three subsequent times with high salt buffer (hypotonic + 1M
NaCl). After final wash, membranes were resuspended in hypotonic buffer
supplemented with 40% glycerol at a volume of 12.5ml per initial liter of culture.
Membranes were flash frozen with liquid nitrogen in 50ml conicals and stored at -80°C.
Frozed membranes were thawed at on ice and volume was raised to 25ml with
hypotonic buffer. Iodoacetimide was dissolved in hypotonic buffer and added at 2mg/ml
final concentration to membranes. Samples were then rotated at 4°C for 30 minutes.
25ml of 2X solubilization buffer (50mM HEPES pH 7.5, 800mM NaCl, 10% glycerol, 2%
MNG, 0.5% CHS) was added to samples and samples were rotated at 4°C for 2.5
hours. Insoluble material was removed by centrifugation at 180,000 x g for 30min.
Solubilized protein was transferred to a 50ml conical and supplemented with 20mM final
imidazole and 0.25ml packed TALON resin (prewashed with water). Samples were
rotated for 16 hours at 4°C. TALON was collected by centrifugation at 700 x g for 5min,
supernatant was discarded, and resin was transferred to a disposable BioRad column
with W1 buffer (50mM HEPES pH 7.5, 400mM NaCl, 25mM imidazole, 10% glycerol,
0.1% MNG, 0.02% CHS). Resin was washed with 10 column volumes of W1 followed
by 5 column volumes of W2 (50mM HEPES pH 7.5, 150mM NaCl, 25mM imidazole,
10% glycerol, 0.01% MNG, 0.002% CHS) and protein was eluted in 5-minute intervals
with 12 x 0.2 column volumes of E buffer (50mM HEPES pH 7.5, 150mM NaCl, 250mM
imidazole, 10% glycerol, 0.01% MNG, 0.002% CHS). 1ul of each elution fraction was
mixed with 4ul of elution buffer and 95ul of Bradford reagent to determine peak
fractions. Four peak fractions were pooled and protein was run over a desalting column
to remove imidazole. Protein was deglycosylated (either PNGase or EndoH from NEB)
72
and treated with 3C protease (Sigma) overnight. Protein was incubated with TALON for
1 hour to remove protease and contaminating proteins. TALON was removed by
spinning sample over disposable BioRad column and concentrated in a 100MWCO
Vivaspin 500.
Yeast nanobody selection
Yeast was thawed and grown in 250ml of -Trp Galactose media for 48hours at 25°C
5x10^9 yeast cells were pelleted and resuspended in 5ml selection buffer (150mM
NaCl, 25mM HEPES pH 7.5, 10% glycerol, 1mM maltose) containing 328nM M2-Flag
and 500ul ms-IgG beads for negative selection. After negative selection yeast were
incubated in 5ml selection buffer containing 500nM ORF74 (Construct ID #208), 328nM
M2-flag and 500ul ms-IgG beads were used for positive selection. After selection yeast
were recovered for 24 hours at 30°C in -Trp glucose media. Round two selection used
5x10^8 cells in 1ml volume. Negative selection was performed with 984nM CXCL1-
ORF74 (Construct ID #207, #210) 656nM M2-Flag and 200ul ms-IgG beads. Positive
selection was performed with 984nM ORF74, 656nM M2-Flag-FITC, and 200ul FITC
beads. Round three selection used 5x10^8 cells in 1ml volume. Negative selection was
performed with 984nM CXCL1-ORF74 656nM M2-Flag-FITC and 200ul FITC beads.
Positive selection was performed with 984nM ORF74, 656nM M2-Flag-CY5, and 200ul
CY5 beads. Round four selection used 5x10^7 cells in 1ml. Cell sorting was used to
isolate yeast labeled with 984nM ORF74 and 656nM M2-Flag-FITC. Cells were sorted
at 0.2% positive about 4x10^5 cells were isolated. Round five was performed as in
73
round four but cells were sorted at 5% positive and about 8x10^5 cells were isolated
and plated on -Trp glucose agar plates to isolate single clones.
Nanobody Purification
pET62b containing nanobody genes was transformed into BL21 cells and grown on LB-
kanamycin plates. Single colonies were used to inoculate 3ml LB containing 0.2%
glucose, 50ug/ml KAN, 37ug/ml chloramphenicol. After 9 hours at 37°C, 3ml culture was
added to 100ml of LB containing 0.2% glucose, 50ug/ml KAN, 34ug/ml chloramphenicol
and grown at 30°C overnight. Overnight culture was used to inocculate 2x250ml at OD
600 = 0.15 in TB containing 0.1% glucose, 50ug/ml KAN, 34ug/ml chloramphenicol, and
1mM MgCl2. Culture was grown at 30°C and monitored until OD 600 was 0.6-0.8 (about
5 hours). 1mM IPTG was added and cells were grown at 25°C for 15 hours, then
pelleted at 4,100 x g for 10min. Cells were resuspend to a final volume of 96ml in cold
TES buffer (200mM Tris-HCl pH 8, 0.5mM EDTA, 500mM Sucrose). 191ml of ice-cold
Milli-q water was added and cells were osmotically shocked with stirring for 45 minutes
at 4°C. Lysate was adjusted to a concentration of 150mM NaCl, 2mM MgCl2, and
20mM imidazole and centrifuged at 20,000 x g for 20min at 4°C. Supernatant was
applied to a gravity column containing 2mL of TALON resin. Resin was washed
sequentially with 3 column volumes 20mM HEPES, pH 7.5, 500mM NaCl, 20mM
Imidazole and 3 column volumes 20mM HEPES, pH 7.5, 100mM NaCl, 20mM
Imidazole. Protein was eluted with 3 column volumes 20mM HEPES, pH 7.5, 100mM
NaCl, 400mM Imidazole. A 3MWCO centrifugal concentrator was used to reduce
sample volume to ~500uL. Sample was desalted into 2x HBS (40mM HEPES pH7.5,
74
200mM NaCl). An equal volume of 100% glycerol was added to sample and after mixing
protein was stored at -20°C. Yield ranged from 20-34mg/L of culture.
Analytical Size Exclusion Chomatography and Gel Filtration
Protein was analyzed by size exclusion chromatography by on either a AdvanceBio
SEC 300A 2.7um 4.6x150mm or Sephadex 300 column run with SEC buffer (150mM
NaCl, 50mM HEPES pH 7.5, .01% MNG, .002% CHS). For gel filtration samples were
run on a Superdex 200 Increase 10/300 GL column with a buffer composed of: 150mM
NaCl, 25mM HEPES pH 7.5, 10% glycerol, .01% MNG, .002% CHS. Samples were
collected from fractions according to absorbance at A280 and used for subsequent
analysis.
Thermalstability Assay
10-15ug of protein with greater than 90% purity was diluted in 150ul of buffer (150mM
NaCl, 25mM HEPES pH 7.5, 10% glycerol, .01% MNG, .002% CHS) containing 10uM
N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]-maleimide (CPM, Molecular Probes
Cat. No. D-346). Solution was incubated in the dark on ice for 30 minutes. Samples are
transferred to quartz cuvettes and loaded into a Cary-Eclipse fluorescent
spectrophotometer. Fluorescence was recorded as temperature from 20-95°C at a rate
of 2°C per minute. Data was imported into Prism (Graphpad) and the melting
temperature was calculated by taking the first derivative of the fluorescence curve.
75
Reconstitution of receptor in lipid cubic phase
20mg of cholesterol was weighed and placed in an amber vial. 180mg of monoolein was
then weighed and combine with cholesterol in the vial. Vial was placed in a fume hood
and 250ul of chloroform was added using a glass Pasteur pipette attached to a P1000
pipette. Cap was placed on vial and gentle tapping of vial was used to mix and dissolve
the powder. The cap was then removed, and the vial was placed under nitrogen for 20
minutes at a flow rate that ensured turbulence on the surface of the dissolved lipids. At
the end of the 20 minutes and once the liquid was relatively stationary, the vial was
covered with aluminum foil containing small perforations and put under vacuum for
approximately 16 hours. Lipids were then stored at -80°C until ready to use.
Lipids and a 100ul gas-tight Hamilton syringe were warmed in a 42°C heat block. Lipids
were transferred from vial to syringe using a P200 pipette with a gel loading tip.
Concentrated protein solution was transferred to a separate gas-tight Hamilton syringe.
The volume of lipids was then adjusted to ensure a 3:2 ratio of lipid volume to protein
volume. A coupler was used to join the two syringes and the protein solution was
injected into the lipid containing syringe. Back and forth mixing was performed until the
solution was optically transparent. The solution was then pushed into one syringe, the
coupler was removed, and a needle was attached for dispensing onto crystallization
plates.
Crystallization Trials
MemMeso and MemGoldMeso crystallization screens were purchased from molecular
dimensions. PEG400, stock options salt kit, and stock options pH kit were purchased
76
from Hampton research and used to set up custom screens detailed in Table 9. An NT8
dropsetter from Formulatrix was used to dispense sample and overlay precipitant
solution on 96 well glass plates. A coverslip was then attached and plates were
incubated at 20°C in a Formulatrix ROCK IMAGER and images for each well were
taken under bright field and cross polarized light at regular intervals.
Table 9 Salt and pH crystallization screens.
Well ID Number Salt Salt Conc Buffer Buffer pH
Buffer
Conc Precipitant
A1 1 Ammonium acetate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A2 2 Ammonium chloride 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A3 3 Ammonium phosphate monobasic 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A4 4 Ammonium fl uoride 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A5 5 Ammonium formate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A6 6 Ammonium citrate dibasic 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A7 7 Ammonium phosphate dibasic 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A8 8 Ammonium nitrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A9 9 Ammonium sulfate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A10 10 Ammonium tartrate dibasic 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A11 11 Calcium acetate hydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
A12 12 Calcium chloride dihydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B1 13 Lithium acetate dihydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B2 14 Lithium chloride 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B3 15 Lithium citrate tribasic tetrahydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B4 16 Lithium nitrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B5 17 Lithium sulfate monohydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B6 18 Magnesium acetate tetrahydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B7 19 Magnesium chloride hexahydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B8 20 Magnesium formate dihydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B9 21 Magnesium nitrate hexahydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B10 22 Magnesium sulfate hydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B11 23 Nickel(II) chloride hexahydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
B12 24 Potassium acetate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C1 25 Potassium chloride 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C2 26 Potassium citrate tribasic monohydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C3 27 Potassium phosphate monobasic 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C4 28 Potassium fl uoride 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C5 29 Potassium formate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C6 30 Potassium phosphate dibasic 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C7 31 Potassium nitrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C8 32 Potassium sodium tartrate tetrahydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C9 33 Potassium sulfate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C10 34 Potassium thiocyanate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
77
C11 35 Sodium acetate trihydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
C12 36 Sodium chloride 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D1 37 Sodium citrate tribasic dihydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D2 38 Sodium phosphate monobasic 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D3 39 Sodium formate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D4 40 Sodium phosphate dibasic dihydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D5 41 Sodium malonate pH 7.0 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D6 42 Sodium nitrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D7 43 Sodium sulfate decahydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D8 44 Sodium tartrate dibasic dihydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D9 45 Sodium thiocyanate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D10 46 Tacsimate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D11 47 Zinc acetate dihydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
D12 48 Zinc sulfate heptahydrate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E1 49 Ammonium acetate 0.1 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E2 50 Ammonium chloride 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E3 51 Ammonium phosphate monobasic 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E4 52 Ammonium fl uoride 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E5 53 Ammonium formate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E6 54 Ammonium citrate dibasic 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E7 55 Ammonium phosphate dibasic 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E8 56 Ammonium nitrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E9 57 Ammonium sulfate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E10 58 Ammonium tartrate dibasic 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E11 59 Calcium acetate hydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
E12 60 Calcium chloride dihydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F1 61 Lithium acetate dihydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F2 62 Lithium chloride 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F3 63 Lithium citrate tribasic tetrahydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F4 64 Lithium nitrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F5 65 Lithium sulfate monohydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F6 66 Magnesium acetate tetrahydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F7 67 Magnesium chloride hexahydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F8 68 Magnesium formate dihydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F9 69 Magnesium nitrate hexahydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F10 70 Magnesium sulfate hydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F11 71 Nickel(II) chloride hexahydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
F12 72 Potassium acetate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G1 73 Potassium chloride 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G2 74 Potassium citrate tribasic monohydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G3 75 Potassium phosphate monobasic 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G4 76 Potassium fl uoride 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G5 77 Potassium formate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G6 78 Potassium phosphate dibasic 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G7 79 Potassium nitrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G8 80 Potassium sodium tartrate tetrahydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G9 81 Potassium sulfate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G10 82 Potassium thiocyanate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G11 83 Sodium acetate trihydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
G12 84 Sodium chloride 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H1 85 Sodium citrate tribasic dihydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
78
H2 86 Sodium phosphate monobasic 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H3 87 Sodium formate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H4 88 Sodium phosphate dibasic dihydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H5 89 Sodium malonate pH 7.0 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H6 90 Sodium nitrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H7 91 Sodium sulfate decahydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H8 92 Sodium tartrate dibasic dihydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H9 93 Sodium thiocyanate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H10 94 Tacsimate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H11 95 Zinc acetate dihydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
H12 96 Zinc sulfate heptahydrate 0.4 M Na Citrate pH 5.0 0.1 M 30% (v/v) PEG400
Cloning and primers
Cloning was performed using the Gibson assembly method and primers used are listed
in the following table. For cloning into pIRES and pEZT, vectors were linearized with
NheI+NotI digest, insert was amplified by PCR and the Gibson method was used to
assemble the fragments. For cloning into pFastBaci, the same method was used but the
digest sites were BamHI+NotI. Point mutations and deletions in ORF74 and CXCL1
were generated by PCR with primers including the mutation Gibson assembly. Other
chemokine constructs were synthesized by IDT as gBlocks to include relevant
mutations and tags and cloned into vectors using Gibson assembly. Nanobody
sequences were PCR amplified from yeast and cloned into periplasm expression vector
pET62b with a C terminal 6 histidine tag.
vMIP-NheI-F TTATgctagcgccATGGACACCAAGGGCA
vMIP-NotI-R AATAgcggccgcAGCATAATCAGGAACATCATACGGATATTAGCGAGCAGTGACT
vMIP-W28C-F GAGCGTCCtgtCATAGACCGGACAAGTGC
vMIP-W28C-R TCTATGacaGGACGCTCCCAGCGTGT
ORF74-NheI-F aattacagctcttaaggcagctagcaccatgaagacgatc
ORF74-NotI-R gggcggaattgggctagagcggccgcctaatgatgatgatg
ORF74-Y197C-F TATGTGCtgcGAGAACGCTGGAA
ORF74-Y197C-R CGTTCTCgcaGCACATAGCCTGCT
CXCL1-T39C-F TCCGTGGCCtgtGAACTGCGCTGCCA
79
CXCL1-T39C-R GCAGTTCacaGGCCACGGACGCTCCT
pCDNA3.1-
CXCL1-NheI-F tatagggagacccaagctgggctagcggatccgccacc
pCDNA3.1-
CXCL1-R agcgggtttaaacgggccctgcggccgcataagcataatc
CXCL1-NheI-F ttatgctagcgccatggcccgcgctg
CXCL1-NotI-R aatagcggccgctcagttggatttgtcac
CXCL1-NheI-F aattacagctcttaaggcagctagcggatccgccaccatggcccgcgctgctctc
CXCL1-HA-NotI-
R gggcggaattgggctagagcggccgctcaagcataatcaggaacatcatacggatat
ORF74-K192C-F TGTGTCCtgtCAGGCTATGTGCT
ORF74-K192C-R TAGCCTGacaGGACACAGGGTCCA
ORF74-Q193C-F GTCCAAGtgtGCTATGTGCTACGA
ORF74-Q193C-R ACATAGCacaCTTGGACACAGGGT
ORF74-A194C-F GTCCAAGCAGtgtATGTGCTACGAGAACG
ORF74-A194C-R GTAGCACATacaCTGCTTGGACACAG
ORF74-M195C-F CAGGCTtgtTGCTACGAGAACGCT
ORF74-M195C-R GTAGCAacaAGCCTGCTTGGACA
ORF74-E198C-F TGTGCTACtgtAACGCTGGAAACATGAC
ORF74-E198C-R TCCAGCGTTacaGTAGCACATAGCCTGCT
ORF74-N199C-F TGCTACGAGtgtGCTGGAAACATGAC
ORF74-N199C-R CATGTTTCCAGCacaCTCGTAGCACATAG
ORF74-A200C-F GAGAACtgtGGAAACATGACCGCCGA
ORF74-A200C-R GTCATGTTTCCacaGTTCTCGTAGCACATAG
vMIP-G25C-F ACACGCTGtgtGCGTCCTGGCATA
vMIP-G25C-R AGGACGCacaCAGCGTGTCCCCAGATT
vMIP-A26C-F CTGGGAtgtTCCTGGCATAGACCGGACAA
vMIP-A26C-R ATGCCAGGAacaTCCCAGCGTGT
vMIP-S27C-F TGGGAGCGtgtTGGCATAGACCGGACAA
vMIP-S27C-R GTCTATGCCAacaCGCTCCCAGCGTGT
vMIP-H29C-F TCCTGGtgtAGACCGGACAAGTGCT
vMIP-H29C-R TCCGGTCTacaCCAGGACGCT
vMIP-R30C-F TGGCATtgtCCGGACAAGTGCTGTCT
vMIP-R30C-R TGTCCGGacaATGCCAGGACGCT
vMIP-P31C-F TGGCATAGAtgtGACAAGTGCTGTCTC
vMIP-P31C-R ACTTGTCacaTCTATGCCAGGACGCT
ORF74-S93C-F atctgcctgaacTGTctgtgcctgtcca
ORF74-S93C-R tggacaggcacagACAgttcaggcagat
ORF74-A78C-F ATCACGCtgtGGAGCCATCGACATCCT
80
ORF74-A78C-R ATGGCTCCacaGCGTGATCTGTGCTTG
ORF74-Q323C-F TGTTCCGCtgtCGTATGTACGGACT
ORF74-Q323C-R GTACATACGacaGCGGAACAGGGAG
ORF74-
323QRM325-
AAA-F TGTTCCGCgctgcagctTACGGACTGTTCCAGT
ORF74-
323QRM325-
AAA-R AGTCCGTAagctgcagcGCGGAACAGGGA
vMIP-HA-NotI-R
AATAgcggccgcAGCATAATCAGGAACATCATACGGATAGCGAGCAGTGACTGGTAA
TTG
CXCL1-S36C-F AGGAGCGtgtGTGGCCACTGAACTGCGCT
CXCL1-S36C-R TTCAGTGGCCACacaCGCTCCTGCTGCG
CXCL1-V37C-F AGCGTCCtgtGCCACTGAACTGCGCT
CXCL1-V37C-R TTCAGTGGCacaGGACGCTCCTGCTGC
CXCL1-A38C-F AGCGTCCGTGtgtACTGAACTGCGCTGCCAGT
CXCL1-A38C-R AGTTCAGTacaCACGGACGCTCCTGCT
CXCL1-E40C-F TGGCCACTtgtCTGCGCTGCCAGTGCTT
CXCL1-E40C-R AGCGCAGacaAGTGGCCACGGACGCTCCTGCT
CXCL1-L41C-F CACTGAAtgtCGCTGCCAGTGCTTGCAGA
CXCL1-L41C-R ACTGGCAGCGacaTTCAGTGGCCACGGA
CXCL1-R42C-F TGAACTGtgtTGCCAGTGCTTGCAGA
CXCL1-R41C-R ACTGGCAacaCAGTTCAGTGGCCACGGA
CXCL1-E40C-F2 GCCACTTGTCTGCGCTGCCAGTGCTTGCA
ORF74-Q323C-
F2 TGTTCCGCtgtCGTATGTACGGACTGTTCCAGT
ORF74-Q323C-
R2 AGTCCGTACATACGcacGCGGAACAGGGA
vMIP-Gibson-
NheI-F tatagggagacccaagctggGCTAGCGCCATGGACACC
vMIP-Gibson-
NotI-R cagatatccagcacagtggcgcggccgctcaAGCATAATCAGGAACATCATACGGATAG
ORF74-
323QRM325-
AAA-F2 TGTTCCGCgctgcagctTACGGACTGTTCCAGTCCCTGCGT
ORF74-
323QRM325-
AAA-R AGTCCGTAagctgcagcGCGGAACAGGGAGCCCAGGCAT
CXCL1-Q50C-F AGACCCTGtgtGGAATTCACCCCAAGA
CXCL1-Q50C-R GAATTCCacaCAGGGTCTGCAAGCACT
CXCL1-G51C-F CTGCAGtgtATTCACCCCAAGAACATC
CXCL1-G51C-R GTGAATacaCTGCAGGGTCTGCAAGCA
CXCL1-I52C-F TGCAGGGAtgtCACCCCAAGAACATCCAAAGT
CXCL1-I51C-R TTGGGGTGacaTCCCTGCAGGGTCTGCA
81
CXCL1-H53C-F AGGGAATTtgtCCCAAGAACATCCAAAGTGT
CXCL1-H53C-R TCTTGGGacaAATTCCCTGCAGGGTCT
CXCL1-P54C-F CAGGGAATTCACtgtAAGAACATCCAAAGTGTGA
CXCL1-P54C-R GATGTTCTTacaGTGAATTCCCTGCAGGGT
CXCL1-K55C-F ATTCACCCCtgtAACATCCAAAGTGTGAAC
CXCL1-K55C-R TGGATGTTacaGGGGTGAATTCCCTGCAG
CXCL1-N56C-F ACCCCAAGtgtATCCAAAGTGTGAACGTGA
CXCL1-N56C-R ACTTTGGATacaCTTGGGGTGAATTCCCTGCA
CXCL1-G81C-F CAAGAATtgtCGGAAAGCTTGCCTCAATCCT
CXCL1-G81C-R AGCTTTCCGacaATTCTTGAGTGTGGCTATGA
CXCL1-R82C-F AATGGGtgtAAAGCTTGCCTCAATCCTGCA
CXCL1-R82C-R AGGCAAGCTTTacaCCCATTCTTGAGTGTGGCT
CXCL1-K83C-F AAGAATGGGCGGtgtGCTTGCCTCAATCCT
CXCL1-K83C-R GAGGCAAGCacaCCGCCCATTCTTGAGTGT
CXCL1-A84C-F ATGGGCGGAAAtgtTGCCTCAATCCTGCATC
CXCL1-A84C-R ATTGAGGCAacaTTTCCGCCCATTCTTGAGTG
ORF74-G25C-F GAACATGTCTtgtTACGACTACTCCGGTAACTTC
ORF74-G25C-R GAGTAGTCGTAacaAGACATGTTCAGAGTTTCG
ORF74-Y26C-F TGTCTGGAtgtGACTACTCCGGTAACTTCAGC
ORF74-Y26C-R AGTAGTCacaTCCAGACATGTTCAGAGTTTCG
ORF74-D27C-F CTGGATACtgtTACTCCGGTAACTTCAGCCTG
ORF74-D27C-R TACCGGAGTAacaGTATCCAGACATGTTCAGA
ORF74-Y28C-F TGGATACGACtgtTCCGGTAACTTCAGCCT
ORF74-Y28C-R TTACCGGAacaGTCGTATCCAGACATGTTCAG
ORF74-S29C-F GGATACGACTACtgtGGTAACTTCAGCCTGGA
ORF74-S29C-R TGAAGTTACCacaGTAGTCGTATCCAGACATG
ORF74-G30C-F CTACTCCtgtAACTTCAGCCTGGAGGTGTCT
ORF74-G30C-R CTGAAGTTacaGGAGTAGTCGTATCCAGACATG
ORF74-N31C-F TACTCCGGTtgtTTCAGCCTGGAGGTGTCTG
ORF74-N31C-R CCAGGCTGAAacaACCGGAGTAGTCGTATCCA
ORF74-F32C-F TCCGGTAACtgtAGCCTGGAGGTGTCTGTCT
ORF74-F32C-R CCAGGCTacaGTTACCGGAGTAGTCGTATCCA
ORF74-G25C-F2 GAACATGTCTtgtTACGACTACTCCGGTAACTTCAGCT
ORF74-V36C-F TTCAGCCTGGAGtgtTCTGTCTGCGAAATGACCA
ORF74-V36C-R TCGCAGACAGAacaCTCCAGGCTGAAGTTACCGGA
ORF74-S37C-F ACTTCAGCCTGGAGGTGtgtGTCTGCGAAATGACCA
ORF74-S37C-R TGGTCATTTCGCAGACacaCACCTCCAGGCTGAAGT
82
ORF74-V38C-F AGGTGTCTtgtTGCGAAATGACCACTGTGGT
ORF74-V38C-R GTCATTTCGCAacaAGACACCTCCAGGCTGAAGT
vMIP-NheI-F aattacagctcttaaggcaggctagcgccaccATGGACACCAAGGGCATC
vMIP-Fc-R cacttccggaGCGAGCAGTGACTGGTAATTG
vMIP-Fc-F cactgctcgcTCCGGAAGTGGCAGTGCC
Fc-NotI-R gggcggaattgggctagagcGGCCGCTAATGGTGATGG
vMIP-QQEE-Fc-
R cacttccggagcgagcagtgactggtaattcctcCATCAGCTTCTTCACCCA
CXCL1-Q44C-F ACTGAACTGCGCTGCtgtTGCTTGCAGACCCT
CXCL1-Q44C-R AGGGTCTGCAAGCAacaGCAGCGCAGTTCAGT
CXCL1-L46C-F AACTGCGCTGCCAGTGCtgtCAGACCCTGCAGGGAAT
CXCL1-L46C-R ATTCCCTGCAGGGTCTGacaGCACTGGCAGCGCAGTT
CXCL1-Q47C-F TGCGCTGCCAGTGCTTGtgtACCCTGCAGGGAAT
CXCL1-Q47C-R ATTCCCTGCAGGGTacaCAAGCACTGGCAGCGCA
CXCL1-T48C-F TGCCAGTGCTTGCAGtgtCTGCAGGGAATTCA
CXCL1-T48C-R TGAATTCCCTGCAGacaCTGCAAGCACTGGCA
CXCL1-L49C-F AGTGCTTGCAGACCtgtCAGGGAATTCACCCCAA
CXCL1-L49C-R TTGGGGTGAATTCCCTGacaGGTCTGCAAGCACT
ORF74-E35C-F GGTAACTTCAGCCTGtgtGTGTCTGTCTGCGAAATG
ORF74-E35C-R CATTTCGCAGACAGACACacaCAGGCTGAAGTTACC
CXCL1-Q71C-F GGACCCCACTGCGCCtgtACCGAAGTCATAGCCA
CXCL1-Q71C-R TGGCTATGACTTCGGTacaGGCGCAGTGGGGTCC
CXCL1-L86C-F ATGGGCGGAAAGCTTGCtgtAATCCTGCATCC
CXCL1-L86C-R GGATGCAGGATTacaGCAAGCTTTCCGCCCAT
CXCL1-10His-
Gibson
gggcggaattgggctagagcggccgctaatggtgatggtgatggtggtggtggtgatgAGCATAATCAGGAAC
ATCATACGGATATCTAGAG
ORF74-No10His-
NotI-R aatatGCGGCCGCttaAGTGGTAGCACCTGACATG
ORF74-NoHis-
dC8-NotI-R aatatGCGGCCGCttaCTGACGCAGGGACTGGAAC
CXCL1-K55C-F CAGGGAATTCACCCCtgtAACATCCAAAGTGTG
CXCL1-K55C-R CACACTTTGGATGTTacaGGGGTGAATTCCCTG
CXCL1-I57C-F GAATTCACCCCAAGAACtgtCAAAGTGTGAACGTGA
CXCL1-I57C-R TCACGTTCACACTTTGacaGTTCTTGGGGTGAATTC
CXCL1-Q58C-F TCACCCCAAGAACATCtgtAGTGTGAACGTGAAGT
CXCL1-Q58C-R ACTTCACGTTCACACTacaGATGTTCTTGGGGTGA
ORF74-dN5-
AscI-F TAGTAGggcgcgccgTTCCTGACCATCTTCCTG
ORF74-dN10-
AscI-F TAGTAGggcgcgccgCTGGACGACGACGAGAGCTGGAACGAAA
83
ORF74-dN15-
AscI-F TAGTAGggcgcgccgAGCTGGAACGAAACTCTG
ORF74-dN21-
AscI-F TAGTAGggcgcgccgAACATGTCTGGATACGAC
ORF74-dN25-
AscI-F TAGTAGggcgcgccgTACGACTACTCCGGTAACT
ORF74-dN30-
AscI-F TAGTAGggcgcgccgAACTTCAGCCTGGAGGTGT
ORF74-dN35-
AscI-F TAGTAGggcgcgccgGTGTCTGTCTGCGAAATGA
ORF74-N-dC5-R CCGTACATACGCTGGCGG
ORF74-C-dC5-R ccagcgtatgtacggactgttccagtccctgcgtcagtctttcatgggccggcctctggaagttctgttccaggggcccc
ORF74-No10His-
dC5-NotI-R aatatGCGGCCGCttaCATGAAAGACTGACGCAGGGACT
ORF74-5'-NoBril-
R tggcctgcagCTTAGTACGGCGGACCAC
ORF74-3'-NoBril-
F ccgtactaagCTGCAGGCCAGGAGAAAG
ORF74-L169W-F TGCTGACTTCCGCTGCCtggCTGATCGCTCTGGTGCT
ORF74-L169W-R AGCACCAGAGCGATCAGccaGGCAGCGGAAGTCAGCA
ORF74-R212C-F TGGAGGCTGCACGTCtgtACCGTGAGCGTC
ORF74-R212C-R GACGCTCACGGTacaGACGTGCAGCCTCCA
ORF74-D274C-F TCCTGAACCTGCTGtgtACCCTGCTGCGCCGTA
ORF74-D274C-R TCCTGAACCTGCTGtgtACCCTGCTGCGCCGTA
ORF74-R208C-F TGACCGCCGACTGGtgtCTGCACGTCAGAA
ORF74-R208C-R TTCTGACGTGCAGacaCCAGTCGGCGGTCA
ORF74-D274C-R AGCAGGGTacaCAGCAGGTTCAGGACGTGGTA
ORF74-NoSP-
NoFlag-F aattacagctcttaaggcagctagcaccatgGCTGCCGAGGACTTCCTG
ORF74-NheI-F aattacagctcttaaggcagctagcaccATGAAGACGATCATCGCC
ORF74-dC3-R gaacttccagACCTGACATGAAAGACTG
3C-10His-Stop-F cagtctttcatgtcaggtCTGGAAGTTCTGTTCCAGGGGC
3C-10His-Stop-
NotI-R gggcggaattgggctagagcTTGGGCTAGAGCGGCCGC
pVitroORF74f gtgaaaactacccctaaaagccaGCCACCATGAAGACGATC
pVitroORF74r atcttatcatgtctggccagGCTAGCCTAATGATGATGATG
pVitroCXCL1f aaccggtgatatcggatccaGCCACCATGGCCCGCGCT
pVitroCXCL1r
gtcattggggaaacctgctcACGCGTTCAAGCATAATCAGGAACATCATACGGATATCTAGA
G
pVitroCXCL2f caaagcaaccggtgatatcgGGATCCGCCACCATGGCC
pVitroCXCL2r tcattggggaaacctgctcaacgcgtTCAAGCATAATCAGGAACATCATACGGATATCTAG
pVitroCXCL5f caaagcaaccggtgatatcgGGATCCGCCACCATGTCTC
pVitroCXCL5r tcattggggaaacctgctcaacgcgtTCAAGCATAATCAGGAACATCATACG
84
pVitroCXCL7f caaagcaaccggtgatatcgGGATCCGCCACCATGAGC
pVitroCXCL7r tcattggggaaacctgctcaacgcgtTCAAGCATAATCAGGAACATCATACGG
pVitroCXCL10f caaagcaaccggtgatatcgGGATCCGCCACCATGAAC
pVitroCXCL10r tcattggggaaacctgctcaacgcgtTCAAGCATAATCAGGAACATCATAC
pVitroCXCL12f caaagcaaccggtgatatcgGGATCCGCCACCATGAAC
pVitroCXCL12r tcattggggaaacctgctcaacgcgtTCAAGCATAATCAGGAACATCATAC
ORF74-L170W-F TGACTTCCGCTGCCCTGtggATCGCTCTGGTGCTCTCT
ORF74-L170W-R AGAGAGCACCAGAGCGATccaCAGGGCAGCGGAAGTCA
ORF74-L169-
170W-F TGACTTCCGCTGCCtggtggATCGCTCTGGTGCTCTCT
ORF74-L169-
170W-R AGAGAGCACCAGAGCGATccaccaGGCAGCGGAAGTCA
CXCL1-NheI-F aattacagctcttaaggcagGCTAGCGCCACCATGGCA
CXCL1-
3C3xFlag-XbaI-R gggcggaattgggctagagcTCTAGAGTTACTCTTGTCACTGTTCAGC
CXCL12-NheI-F aattacagctcttaaggcagGCTAGCGCCACCATGAAC
CXCL12-
3C3xFlag-XbaI-R gggcggaattgggctagagcTCTAGACTTGTTTAAAGCTTTCTCCAG
CXCL12-NheI-F aattacagctcttaaggcagGCTAGCGGATCCGCCACC
CXCXL12-NotI-R gggcggaattgggctagagcGCGGCCGCTCAAGCATAATC
CXCL12-K23C-F TGCCTCAGCGACGGGtgtCCCGTCAGCCTGAGC
CXCL12-K23C-R GCTCAGGCTGACGGGacaCCCGTCGCTGAGGCA
CXCL12-P23C-F tcagcgacgggAAGtgtGTCAGCCTGAGCTACA
CXCL12-P23C-R TGTAGCTCAGGCTGACacaCTTcccgtcgctga
CXCL12-V24C-F tcagcgacgggAAGCCCtgtAGCCTGAGCTACAGAT
CXCL12-V24C-R ATCTGTAGCTCAGGCTacaGGGCTTcccgtcgctga
CXCL12-S25C-F acgggAAGCCCGTCtgtCTGAGCTACAGAT
CXCL12-S25C-R ATCTGTAGCTCAGacaGACGGGCTTcccgt
CXCL12-L26C-F acgggAAGCCCGTCAGCtgtAGCTACAGATGCCCAT
CXCL12-L26C-R ATGGGCATCTGTAGCTacaGCTGACGGGCTTcccgt
CXCL12-S27C-F AAGCCCGTCAGCCTGtgtTACAGATGCCCATG
CXCL12-S27C-R CATGGGCATCTGTAacaCAGGCTGACGGGCTT
CXCL12-Y28C-F AAGCCCGTCAGCCTGAGCtgtAGATGCCCATGCCGAT
CXCL12-Y28C-R ATCGGCATGGGCATCTacaGCTCAGGCTGACGGGCTT
CXCL12-R29C-F TCAGCCTGAGCTACtgtTGCCCATGCCGATT
CXCL12-R29C-R AATCGGCATGGGCAacaGTAGCTCAGGCTGA
ORF74-N18Q-F ACGACGAGAGCTGGcaaGAAACTCTGAACATG
ORF74-N18Q-R CATGTTCAGAGTTTCttgCCAGCTCTCGTCGT
ORF74-N22Q-F TGGAACGAAACTCTGcaaATGTCTGGATACGA
85
ORF74-N22Q-R TCGTATCCAGACATttgCAGAGTTTCGTTCCA
ORF74-N31Q-F TACGACTACTCCGGTcaaTTCAGCCTGGAGGTGT
ORF74-N31Q-R ACACCTCCAGGCTGAAttgACCGGAGTAGTCGTA
ORF74-N202Q-F TACGAGAACGCTGGAcaaATGACCGCCGACTGGA
ORF74-N202Q-R TCCAGTCGGCGGTCATttgTCCAGCGTTCTCGTA
ORF74-N18,22Q-
F ACGACGAGAGCTGGcaaGAAACTCTGcaaATGTCTGGATACGA
ORF74-N18,22Q-
R TCGTATCCAGACATttgCAGAGTTTCttgCCAGCTCTCGTCGT
pNAHA-R TGGCTTTTAGGGGTAGTTTTC
p-NAHA-ORF74-
F gtgaaaactacccctaaaagccaGCCACCATGAAGACGATC
p-NAHA-ORF74-
R catcaatgtatcttatcatgGCTAGCCTAATGATGATGATG
pNAHA-F CATGATAAGATACATTGATGAGTTTG
pNAHA-ORF74-
NoSP-F gtgaaaactacccctaaaagccaATGGCTGCCGAGGACTT
pNAHA-CXCL1-
3Flag-BamHI-F caaagcaaccggtgatatcggatccaGCCACCATGGCAAGAGCTG
pNAHA-CXCL1-
3Flag-MluI-R tcattggggaaacctgctcacgcgtTCACTTGTCGTCGTCGTCTTTATAATC
pNAHA-CXCL1-
Flag-BamHI-F caaagcaaccggtgatatcggatccaGCCACCATGGCCCGCGCT
pNAHA-CXCL1-
3Flag-MluI-R
tcattggggaaacctgctcacgcgtTCATCACTTGTCATCGTCGTCCTTGTAGTCTCTAGAGTT
G
pVITRO2-
CXCL1-HA-F aaaactacccctaaaagccaGCCACCATGGCCCGCGCT
pVITRO2-
CXCL1-HA-R
catcaatgtatcttatcatgTCAAGCATAATCAGGAACATCATACGGATATCTAGAGTTGGAT
TTGTCACTG
pVITRO2-
CXCL1-3F-F aaaactacccctaaaagccaGCCACCATGGCAAGAGCTG
pVITRO2-
CXCL1-3F-R catcaatgtatcttatcatgTCACTTGTCGTCGTCGTCTTTATAATC
pVITRO2-
CXCL1-F-R catcaatgtatcttatcatgTCATCACTTGTCATCGTCGTCCTTGTAGTCTCTAGAGTTG
pVITRO2-
CXCL1-F-F aaaactacccctaaaagccaGCCACCATGGCCCGCGCT
ORF74-G30C-
N31Q-F TACGACTACTCCtGTcaaTTCAGCCTGGAGGTGT
ORF74-G30C-
N31Q-R ACACCTCCAGGCTGAAttgACaGGAGTAGTCGTA
ORF74-N18,22Q-
F TGGcaaGAAACTCTGcaaATGTCTGGATACGA
ORF74-N18,22Q-
R TCGTATCCAGACATttgCAGAGTTTCttgCCA
pVitro-g2-orf74-
MluI-f caaagcaaccggtgatatcgacgcgtGCCACCATGAAGACGATC
pVitro-g2-orf74-
NheI-r tcattggggaaacctgctcaggatccCTAATGATGATGATGATGATGATG
86
pVitro-g2-
orf74noF-MluI-f caaagcaaccggtgatatcgacgcgtatgGCTGCCGAGGACTTCCT
pVitro-g2-orf74-
NheI-r2 tcattggggaaacctgctcaggatccCCGCCTAATGATGATGATGATGATGATG
pVITRO-G1-Seq-
F gaagagttaggccagctt
pVITRO-G1-Seq-
R gtttattgcagcttataatg
pVITRO-G2-Seq-
F ctaattctcgggcttct
pVITRO-G2-Seq-
R acgcagtacaaagtgttac
pires-
orf74notags-f aattacagctcttaaggcaggctagccaccATGGCTGCCGAGGACTTC
pires-
orf74notags-r gggcggaattgggctagagcgcggccgctcaAGCACCTGACATGAAAGACTG
pvitroG2-
orf74notags-f aaagcaaccggtgatatcgacgcgtgccaccATGGCTGCCGAGGACTTC
pvitroG2-
orf74notags-r cattggggaaacctgctcaggatcctcaAGCACCTGACATGAAAGACTG
pVITROg1-Flag-F TCTAGAGACTACAAGGACGAC
pVITROg1-Flag-
R TGGTGGCTGGCTTTTAGG
pVITROg1-
CXCL2-F ctacccctaaaagccagccaccaATGGCACGAGCGACATTG
pVITROg1-
CXCL2-R tcgtccttgtagtctctagaATTACTCTTTCCATTCTTGAGCATTTTTTC
pVITROg1-
CXCL3-F ctacccctaaaagccagccaccaATGGCACATGCCACGCTG
pVITROg1-
CXCL3-R tcgtccttgtagtctctagaGTTCGTTGAGCCTTTATTCAGTATTTTCTC
pVITROg1-
CXCL5-F ctacccctaaaagccagccaccaATGTCTCTTTTGTCCAGTC
pVITROg1-
CXCL5-R tcgtccttgtagtctctagaATTCTCTTTGTTCCCGCC
pVITROg1-
CXCL7-F ctacccctaaaagccagccaccaATGTCTCTCAGACTCGAC
pVITROg1-
CXCL7-R tcgtccttgtagtctctagaATCTGCGGATTCGTCACC
pVITROg1-
CXCL8-F ctacccctaaaagccagccaccaATGACCTCCAAATTGGCG
pVITROg1-
CXCL8-R tcgtccttgtagtctctagaATTCTCAGCCCTCTTCAAG
pVITROg1-
CXCL10-F ctacccctaaaagccagccaccaATGAACCAAACGGCTATTTTG
pVITROg1-
CXCL10-R tcgtccttgtagtctctagaTGGAGATCTCTTGGAGCG
pVITROg1-
CXCL12-F ctacccctaaaagccagccaccaATGAATGCTAAAGTTGTGG
pVITROg1-
CXCL12-R tcgtccttgtagtctctagaCTTATTAAGGGCCTTTTCG
pNAHA-g2-F GTTCAAAAAACTCCTTGTTTC
87
pNAHA-g2-R TACGCGTGGATCCGATATC
pNAHA-g2-
orf74nt-F tgatatcggatccacgcgtaGCCACCATGGCTGCCGAG
pNAHA-g2-
orf74nt-R aaacaaggagttttttgaacTCAAGCACCTGACATGAAAGACTGAC
pVITRO-NOg1-R ggtggcGCTAGCTGGCTTTTAGGG
pVITRO-NOg1-F CATGATAAGATACATTGATGAGTTTG
pVITRO-g2-HA-
ORF74-F
aaagcaaccggtgatatcgagccaccatgtatccgtatgatgttcctgattatgctGCTGCCGAGGACTTCCT
G
ORF74-NoBRIL-
N-R tggcctgcagCTTAGTACGGCGGACCAC
ORF74-NoBRIL-
C-F ccgtactaagCTGCAGGCCAGGAGAAAG
pVITRO-g2-HA-
ORF74-R cattggggaaacctgctcagctatgtagtAGCACCTGACATGAAAGAC
ORF74-W169L-F TGCTGACTTCCGCTGCCctgCTGATCGCTCTGGTGCT
ORF74-W169L-R AGCACCAGAGCGATCAGcagGGCAGCGGAAGTCAGCA
ORF74-E320L-F TACTCATGCCTGGGCTCCctgTTCCGCCAGCGTATGTACGGA
ORF74-E320L-R TCCGTACATACGCTGGCGGAAcagGGAGCCCAGGCATGAGTA
pVg1-CXCL1-3C-
10HIS-F accatcaccatcaccattagCATGATAAGATACATTGATGAGTTTG
pVg1-CXCL1-3C-
10HIS-R ggccatggtggcggcgcgccCTGGCTTTTAGGGGTAGTTTTC
pIRES-CXCL1-
3C-10HIS-F aattacagctcttaaggcagGGCGCGCCGCCACCATGG
pIRES-CXCL1-
3C-10HIS-R gggcggaattgggctagagcgcggccgCTAATGGTGATGGTGATGGTGGTGGTGGTGATG
pvitroG2-
orf74notags-r2 cattggggaaacctgctcaggatcctcaAGTGGTAGCACCTGACATGAAAGACTG
pvitroG2-
ORF74yfp-f aaagcaaccggtgatatcgaacgcgtgccACCATGGCTGCCGAGGAC
pvitroG2-
ORF74yfp-r tgctcaccatGGGCCCCTGGAACAGAAC
pvitroG2-yfp-f ccaggggcccATGGTGAGCAAGGGCGAG
pvitroG2-yfp-r cattggggaaacctgctcagggatcctcaCTTGTACAGCTCGTCCATGC
pEZT-BM-
CXCL1-3C10HIS-
F ttaatacgactcactataggGCCACCATGGCCCGCGCT
pEZT-BM-
CXCL1-3C10HIS-
R cctcggatccctgcagtcgcCTAATGGTGATGGTGATGGTGGTGGTGGTG
pEZT-BM-
ORF74notag-F ttaatacgactcactataggGCCACCATGGCTGCCGAG
pEZT-BM-
ORF74notag-R cctcggatccctgcagtcgcagtggtAGCACCTGACATGAAAGACTGACG
pEZT-BM-
ORF74-YFP-F ttaatacgactcactataggGCCACCATGGCTGCCGAG
pEZT-BM-
ORF74-YFP-R cctcggatccctgcagtcgcTCACTTGTACAGCTCGTCCATGC
88
pIRES-
ORF74yfp-F tacaaggacgatgatgacggcgcgccGGCTGCCGAGGACTTCCT
pIRES-
ORF74yfp-R gggcggaattgggctagagcTCACTTGTACAGCTCGTCC
pIRES-
ORF74yfp-dN35-
F tacaaggacgatgatgacggcgcgccGTGTCTGTCTGCGAAATG
pFastBac-
ORF74notags-F ccgtcccaccatcgggcgcgGCCACCATGGCTGCCGAG
pFastBac-
ORF74notags-R tcctctagtacttctcgacaGCTCAGGATCCTCAAGCACC
pFastBac-
CXCL1-3c10his-F ccgtcccaccatcgggcgcgGCCACCATGGCCCGCGCT
pFastBac-
CXCL1-3c10his-
R tcctctagtacttctcgacaCTAATGGTGATGGTGATGGTGGTGGTGGTG
pEZT-BM-
ORF74notag-F2 ttaatacgactcactatagggccaccatgGCTGCCGAGGACTTCCTG
pEZT-BM-
ORF74notag-R2 cctcggatccctgcagtcgctcaAGTGGTAGCACCTGACATG
pEZT-ORF74-
Cflag-R cctcggatccctgcagtcgctcacttgtcatcgtcgtccttgtagtcAGCACCTGACATGAAAGACTGACG
pEZT-CXCL7-F gactcactatagggccaccaGCCACCATGTCTCTCAGAC
pEZT-CXCL7-R cctggaacagaacttccaggctagcATCTGCGGATTCGTCACC
pEZT-CXCL12-F gactcactatagggccaccaGCCACCATGAATGCTAAAG
pEZT-CXCL12-R cctggaacagaacttccaggctagcCTTATTAAGGGCCTTTTCG
pEZT-Gibson-F GCTAGCCTGGAAGTTCTGTTC
pEZT-Gibson-R TGGTGGCCCTATAGTGAG
pEZT-CXCL7-F taatacgactcactataggctcgagGCCACCATGTCTCTCAGAC
pEZT-CXCL7-R cctggaacagaacttccaggctagcATCTGCGGATTCGTCACC
pEZT-CXCL1-
dC5-F taatacgactcactataggcGCCACCATGGCCCGCGCT
pEZT-CXCL1-
dC5-R
cctggaacagaacttccaggctagcCAGCATCTTTTCGATGATTTTCTTAACTATGGGGGATG
CAGG
pEZT-CXCL2-F taatacgactcactataggcGCCACCATGGCCCGCGCC
pEZT-CXCL2-R
cctggaacagaacttccaggctagcGTTGGATTTGCCATTTTTCAGCATCTTTTCGATGATTTT
CTTAACCATGGGC
pEZT-CXCL3-F taatacgactcactataggcGCCACCATGGCACATGCC
pEZT-CXCL3-R cctggaacagaacttccaggctagcGTTCGTTGAGCCTTTATTCAGTATTTTCTCTATG
pEZT-CXCL4-F taatacgactcactataggcGCCACCATGAGTTCAGCC
pEZT-CXCL4-R cctggaacagaacttccaggctagcAGATTCAAGCAATTTTTTGATGATCTTC
pEZT-CXCL5-F taatacgactcactataggcGCCACCATGTCTCTTTTG
pEZT-CXCL5-R cctggaacagaacttccaggctagcATTCTCTTTGTTCCCGCC
pEZT-CXCL6-F taatacgactcactataggcGCCACCATGTCCCTCCCT
89
pEZT-CXCL6-R cctggaacagaacttccaggctagcGTTTTTCTTATTACCTGAGTCCAATATCTTCTGTATG
pEZT-CXCL8-F taatacgactcactataggcGCCACCATGACCTCCAAATTG
pEZT-CXCL8-R cctggaacagaacttccaggctagcATTCTCAGCCCTCTTCAAGAATTTTTC
pEZT-CXCL10-F taatacgactcactataggcGCCACCATGAACCAAACG
pEZT-CXCL10-R cctggaacagaacttccaggctagcTGGAGATCTCTTGGAGCG
pEZT-vMIPII-F taatacgactcactataggcGCCACCATGGACACAAAGGG
pEZT-vMIPII-R cctggaacagaacttccaggctagcGCGAGCAGTCACCGGCAG
ORF74-T4L-R cgaagatgttAGCAGACTTAGTACGGCGGAC
T4L-F taagtctgctAACATCTTCGAGATGCTG
T4L-R gcagagctgaGTAAGCGTCCCAAGTTCC
ORF74-T4L-F ggacgcttacTCAGCTCTGCAGGCCAGG
ORF74-RUBR-R acttcttcatAGCAGACTTAGTACGGCGGAC
RUBR-F taagtctgctATGAAGAAGTACACCTGCAC
RUBR-R gcagagctgaTTCCTCCACTTCCTCGAAC
ORF74-RUBR-F agtggaggaaTCAGCTCTGCAGGCCAGG
pFastBac-
ORF74flag-R tcctctagtacttctcgacaTCACTTGTCATCGTCGTCC
pFastBac-
ORF74flag-R2 tcctctagtacttctcgacatcacttgtcatcgtcgtccttgtagtcAGCACCTGACATGAAAGACTGACG
pFastBac-
CXCL2-F ccgtcccaccatcgggcgcgGCCACCATGGCCCGCGCC
pFastBac-
CXCL3-F ccgtcccaccatcgggcgcgGCCACCATGGCACATGCC
pFastBac-
CXCL4-F ccgtcccaccatcgggcgcgGCCACCATGAGTTCAGCC
pFastBac-
CXCL5-F ccgtcccaccatcgggcgcgGCCACCATGTCTCTTTTG
pFastBac-
CXCL6-F ccgtcccaccatcgggcgcgGCCACCATGTCCCTCCCT
pFastBac-
CXCL7-F ccgtcccaccatcgggcgcgGCCACCATGTCTCTCAGAC
pFastBac-
CXCL8-F ccgtcccaccatcgggcgcgGCCACCATGACCTCCAAATTG
pFastBac-
CXCL10-F ccgtcccaccatcgggcgcgGCCACCATGAACCAAACG
pFastBac-
CXCL12-F ccgtcccaccatcgggcgcgGCCACCATGAATGCTAAAGTT
pFastBac-vMIPII-
F ccgtcccaccatcgggcgcgGCCACCATGGACACAAAGGG
pFastBac-
CXCL2-F accatcgggcgcggccaccaATGGCACGAGCGACATTG
pFastBac-
CXCL2-R cctggaacagaacttccaggATTACTCTTTCCATTCTTGAGCATTTTTTC
pFB-ORF74-
Flag3C10HIS-R acagaacttccagaggccggcccttgtcatcgtcgtccttgtagtcAGCACCTGACATGAAAGACTGACG
90
ORF74-PGS-R aatcaatgccAGCAGACTTAGTACGGCGGAC
PGS-F taagtctgctGGCATTGATTGTAGCTTCTG
PGS-R gcagagctgaCGAGAACGACATTGCCCTCT
ORF74-PGS-F gtcgttctcgTCAGCTCTGCAGGCCAGG
ORF74-XYL-R cagtgctagcAGCAGACTTAGTACGGCGGAC
XYL-F taagtctgctGCTAGCACTGATTACTGGCA
XYL-R gcagagctgaCCACACAGTCACGTTGCTAG
ORF74-XYL-F gactgtgtggTCAGCTCTGCAGGCCAGG
pFB-ORF74-
NoTag-R2 tcctctagtacttctcgacaAGCACCTGACATGAAAGACTGACG
pFB-IgGk-SP-F ccgtcccaccatcgggcgcgctagcgccaccatggagacg
pFB-FC-R tcctctagtacttctcgacaAGTCGCGCGGCCGCTAATGGTGA
Linker-3C-F TGAGCTCGAGCGGCAGCGGCctcgagGTTCTGTTCCAGGGCCCCGA
Linker-3C-R TCGGGGCCCTGGAACAGAACctcgagGCCGCTGCCGCTCGAGCTCA
pEZT-IgGk-SP-F ACTTAATACGACTCACTATAGGCTAGCGCCACCATGGAGACGGACACA
pEZT-10His-R cctcggatccctgcagtcgctcgtctttgtagtcctaatg
pF-Strep-NheI-
ORF74-F cgtcccaccatcgggcgcgggccaccATGGCTGCCGAGGACTTC
pF-Strep-BamHI-
ORF74-R gaacagaacttccagggatccAGCACCTGACATGAAAGACTG
pF-Strep-NheI-
Nb-F cgtcccaccatcgggcgcgggccaccATGGAGACGGACACACTG
pF-Strep-BamHI-
Nb-R gaacagaacttccagggatccCACGGTCACCTGGGTGCCCT
pEZT-NheI-
ORF74-F ttaatacgactcactatagggccaccATGGCTGCCGAGGACTTC
pEZT-NheI-Nb-F ttaatacgactcactatagggccaccATGGAGACGGACACACTG
91
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Addendum: Post-translational modification of HOIP blocks
TLR4-mediated linear ubiquitin chain formation
This section is adapted from a paper published in 2015 in mBio 01777-15
Authors:
James Bowman
a
, Mary A. Rodgers
a, b
, Mude Shi
a
, Rina Amatya
a
, Bruce Hostager
c
,
Kazuhiro Iwai
d
, Shou-Jiang Gao
a
and Jae U. Jung
a,e,*
a
Department of Molecular Microbiology and Immunology, Keck School of Medicine, Los
Angeles, California, USA
b
Abbott Diagnostics, Abbott Park, Illinois, USA
c
Department of Pediatrics, University of Iowa, Iowa City, Iowa, USA
d
Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto
University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, Japan
e
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy,
University of Southern California, Los Angeles, California, USA
96
Highlights
• LUBAC catalytic subunit HOIP is ubiquitinated at lysine-1056 upon bacterial
lipopolysaccharide (LPS)-mediated TLR4 stimulation
• Ubiquitination of HOIP dynamically alters its conformation and suppresses
LUBAC activity
• HOIP Lys1056®Arg mutation leads to persistent LUBAC activity and
prolonged NF-kB activation
Abstract
Linear ubiquitination is an atypical post-translational modification catalyzed by the
linear ubiquitin chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and
Sharpin. LUBAC facilitates NF-kB activation and inflammation upon receptor
stimulation by ligating linear ubiquitin chains to critical signaling molecules.
Indeed, linear ubiquitination dependent signaling is essential to prevent pyogenic
bacterial infections that can lead to death. While linear ubiquitination is essential
for intracellular receptor signaling upon microbial infection, this response must be
metered and stopped to avoid tissue damage and autoimmunity. While LUBAC is
activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in
response to bacterial stimuli have remained elusive. We demonstrate that LUBAC
activity itself is downregulated through ubiquitination, specifically of the catalytic
subunit HOIP at the carboxyl terminal lysine-1056. Ubiquitination of Lys1056
dynamically altered HOIP conformation, resulting in the suppression of its catalytic
activity. Consequently, HOIP Lys1056®Arg mutation led not only to persistent
LUBAC activity but also to prolonged NF-kB activation induced by bacterial
lipopolysaccharide mediated TLR4 stimulation, whereas it showed no effect on NF-
kB activation induced by CD40 stimulation. This study describes a novel post-
translational regulation of LUBAC-mediated linear ubiquitination that is critical for
specifically directing TLR4-mediated NF-kB activation.
97
Importance
Post-translational modification of proteins enables cells to respond quickly to
infections and immune stimuli in a tightly controlled manner. Specifically, covalent
modification of proteins with the small protein ubiquitin is essential for cells to
initiate and terminate immune signaling in response to bacterial and viral infection.
This process is controlled by ubiquitin ligase enzymes, which themselves must be
regulated to prevent persistent and deleterious immune signaling. However, how
this regulation is achieved is poorly understood. This paper reports a novel
ubiquitination event of the atypical ubiquitin ligase HOIP that is required to
terminate bacterial lipopolysaccharide (LPS)-induced TLR4 immune signaling.
Ubiquitination causes the HOIP ligase to undergo a conformational change, which
blocks its enzymatic activity and ultimately terminates LPS-induced TLR4
signaling. These findings provide a new mechanism for controlling HOIP ligase
activity that is vital to properly regulate a pro-inflammatory immune response.
98
Introduction
Mutations in genes required for regulating anti-microbial or inflammatory signaling
frequently lead to auto-inflammatory diseases and impair the immune system’s ability to
fight infection (1,2). In particular, mounting evidence indicates that proper functioning of
the immune signaling regulatory complex called the linear ubiquitin chain assembly
complex (LUBAC), consisting of heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1L), HOIL-
1L interacting protein (HOIP), and SHANK-associated RH domain interactor (Sharpin), is
required for maintenance of a functional immune system (3,4). In humans and mice,
mutations in LUBAC result in deregulation of immune signaling. For example, human
patients with null mutations in the immunoregulatory RBCK1 gene, which codes for the
protein HOIL-1L, exhibit a combined autoinflammatory/immunodeficient phenotype
characterized by elevated serum levels of pro-inflammatory cytokine IL-6 and chronic
invasive bacterial infections that ultimately results in death (1). In accordance with
immunodeficiency seen in humans, HOIL-1L deficient mice are resistant to LPS-induced
lethal inflammation, suggesting that HOIL-1L is required to mount a systemic innate
immune inflammatory response to bacterial infection (5). In mice, null mutations in
SHARPIN cause chronic proliferative dermatitis as well as eosinophilic inflammation and
impaired lymphoidogenesis (6). Genetic deletion of HOIP is embryonic lethal, however
the B-cell specific ablation of HOIP results in defective antibody responses to thymus-
dependent and independent antigens in mice (7). Additionally, mutations in RNF31, the
gene encoding for HOIP, alter LUBAC activity and are enriched in certain B-cell
lymphomas in humans (8). Collectively, genetic studies have demonstrated that LUBAC
is required for numerous anti-microbial and inflammatory pathways, including NLRP3, IL-
1R, TLR4, TLR2, TNF, NOD2, and CD40 (5, 7, 9-12). Thus, the tight regulation of LUBAC
activity is necessary to maintain a healthy immune response. Aberrant LUBAC activity
contributes to the development of diseases resulting from autoinflammation and
immunodeficiency.
LUBAC function is critical for NF-κB activation, during which it attaches linear
ubiquitin to multiple molecules. The first-characterized function of LUBAC was linear
ubiquitination of the NF-κB essential modulator (NEMO); which is required for NF-κB
activation (11). During TNF-α mediated NF-κB activation, RIP1 is modified with linear
99
ubiquitin in the TNF receptor-signaling complex (6). In contrast to direct substrate
modification, during IL-1R and TLR1/2-mediated NF-κB activation, LUBAC conjugates
linear ubiquitin to existing lysine-63 linked ubiquitin chains on IRAK1, IRAK4, and MyD88
(13). In addition to NF-κB activation, LUBAC mediates induction of other host immune
signaling pathways, including inflammasome activation, by attaching linear ubiquitin
chains to key signaling molecules. During NLRP3 activation, LUBAC modifies the adaptor
protein ASC, promoting ASC/NLRP3 binding and oligomerization (5). This directly leads
to secretion of inflammatory cytokines. Thus, LUBAC facilitates signaling by modifying
substrates, both directly and indirectly, to acutely modulate innate immunity.
LUBAC is a trimeric complex composed of Sharpin, HOIL-1L, and HOIP, where
HOIP is the primary E3 ubiquitin ligase and the only enzyme known to catalyze linear
ubiquitin chain conjugation. HOIP is a member of the RING-in-between-RING (RBR)
family of E3 ubiquitin ligase. Interestingly, RBR ligases often exist in autoinhibited
conformations, requiring protein-protein interactions or post-translational modifications for
conformational change and activation (14). For instance, post-translational modifications
of the well-studied RBR ligase Parkin can both positively and negatively regulate its
activity through altering protein conformation (15, 16). In fact, mutations that enhance
HOIP and HOIL-1L binding, and thus increase LUBAC enzymatic activity, are associated
with human disease (8), suggesting that the strict negative regulation of linear ubiquitin
chain formation is required for the maintenance of immune homeostasis.
Upon stimulation of immune receptors, including TNFα, TLR4 and CD40 receptors,
LUBAC enzymatic activity is rapidly activated and subsequently inactivated through
previously uncharacterized mechanisms. We report that the enzymatically critical LUBAC
subunit HOIP is modified by ubiquitin at multiple lysine residues. Targeted mutations to
block HOIP ubiquitination revealed that a lysine-1056 to arginine (K1056R) mutation
within the carboxyl terminal linear ubiquitin chain determining domain (LDD) resulted in
elevated HOIP activity. TLR4 stimulation induced HOIP ubiquitination, which altered
HOIP conformation and decreased LUBAC activity. Furthermore, blocking HOIP
ubiquitination with the K1056R mutation resulted in elevated and persistent expression of
NF-κB gene expression. Our results suggest that the carboxyl terminal ubiquitination of
HOIP inactivates LUBAC activity upon TLR4 stimulation to prevent persistent NF-κB
100
activation, indicating conformational rearrangement as a primary regulatory mechanism
for RBR ligases.
Results
Identification of HOIP ubiquitination sites
To investigate a role for post-translational modifications in controlling HOIP function, full
length HOIP was purified from 293T cells under denaturing conditions and mass
spectrometry was used to identify ubiquitinated residues. Two residues, lysine-640 and
lysine-1056, were assigned with high confidence as being modified covalently by ubiquitin
(Fig. 1A). Mass spectrometry also identified various ubiquitin peptides corresponding to
linear, lysine-11, lysine-48, and lysine-63 linked ubiquitin chains co-migrating with HOIP.
Specific ubiquitin chain linkages can have different functions when attached to substrate
proteins. Among their functions, lysine-11 and lysine-48 ubiquitin chains can lead to
protein degradation, while methionine-1 and lysine-63 can regulate signal transduction
(17). All four of these ubiquitin chain types have been implicated in LUBAC-mediated
signaling pathways (6). To verify that the lysine-640 and lysine-1056 residues are sites
for HOIP ubiquitination, mutant HOIP constructs were generated where the identified
lysine residues were mutated to arginine and those mutants were then transfected in
293T cells to examine their respective HOIP ubiquitination levels (Fig. 1B). A previously
identified HOIP ubiquitination site was mutated as a positive control (K330R) (18, 19) and
the enzymatically inactive CS (C885S) mutant that is incapable of linear ubiquitination or
autoubiquitination was included as a negative control. The HOIP K330R and K1056R
mutants both showed reduced ubiquitination levels relative to WT. The K640R and CS
mutants did not show altered HOIP ubiquitination levels, indicating that these mutations
did not affect HOIP ubiquitination, and that autoubiquitination is not the primary source of
ubiquitin attached to HOIP under these conditions. Collectively, this demonstrates that
HOIP is ubiquitinated at multiple residues, and that ubiquitination of HOIP does not
require the catalytic C885 residue.
Interestingly, the K1056 residue is highly evolutionarily conserved among
vertebrates and is located in the carboxyl LDD of HOIP that is essential for the protein’s
101
enzymatic activity (Fig. 1A) (20). Specifically, the crystal structure of the HOIP enzymatic
region revealed that the K1056 residue is located on an exposed surface (Fig. S1A) (21).
As HOIP has already been shown to be ubiquitinated at multiple sites in its amino
terminus (19), we generated the carboxyl terminal enzymatic domain constructs of HOIP
and found that the K1056 is the primary site for ubiquitination of the HOIP C-terminus
(Fig. S1B-C). We next tested whether the HOIP C-terminus was capable of
autoubiquitination by performing an in vitro ubiquitination assay using recombinant HOIP
C-terminus. No high molecular weight HOIP bands were detected in this assay,
suggesting that HOIP C-terminus is not efficiently autoubiquitinated in vitro (Fig. S1D). To
investigate the ubiquitin chain linkage at the K1056 of HOIP, we used ubiquitin WT, K48R
or K63R mutant. While both ubiquitin mutants could still ubiquitinate HOIP, neither was
as efficient as ubiquitin WT (Fig. 1C). Next, we used the K48-only or K63-only ubiquitin
mutant where all the lysines except for the K48 or the K63 have been mutated to arginine.
Neither K48-only nor K63-only ubiquitin was capable of efficiently ubiquitinating HOIP
(Fig. 1D). Finally, we used ubiquitin-specific antibodies to compare the ubiquitin linkages
on the C-terminal HOIP-WT and HOIP-K1056R (22). HOIP-WT was modified primarily by
M1, K48, and K63-linked ubiquitin chains, which were among the same linkages identified
by mass spectrometry (Fig. 1E). In contrast, the HOIP-K1056R mutant had dramatically
reduced levels of all types of ubiquitin linkages observed on HOIP-WT (Fig. 1E, Fig. S1E),
suggesting that K1056 is the primary site for ubiquitination in the carboxyl terminus of
HOIP. Finally, to rule out the possibility that the decreased ubiquitination of the HOIP
K1056R mutant occurs because mutating K1056 disrupts HOIP structure and renders
HOIP non-functional, we examined its enzymatic activity in vitro. We purified full length
HOIP-WT and HOIP-K1056R from bacteria using dual expression constructs carrying
both HOIL-1L and HOIP. HOIP-K1056R had equivalent in vitro activity to HOIP-WT,
indicating that this mutation does not alter the intrinsic enzymatic activity of HOIP (Fig.
S1F). These experiments collectively demonstrate that HOIP is ubiquitinated at the K1056
by multiple types of ubiquitin chains and a K1056R mutation disrupts HOIP ubiquitination
without disrupting its intrinsic enzymatic activity.
102
LUBAC kinetics and function in B-cells
We next established a system to study the function of HOIP-K1056 ubiquitination in the
context of immune receptor stimulation. To measure the kinetics of LUBAC activation and
inactivation, we used the A20.2J mouse B-cell lymphoma cell line, which has previously
been used to characterize LUBAC-dependent immune signaling (10). A20 cells were
treated with LPS to activate TLR4, which requires HOIP enzymatic activity for efficient
downstream signaling (7). To measure LUBAC activation, we examined linear
ubiquitination before and after TLR4 activation by purifying linear ubiquitin chains from
cells using two approaches. In the first approach, linear ubiquitin chains were purified
from cells lysed under denaturing conditions using a linear ubiquitin-specific antibody. In
the second approach, linear ubiquitin chains were purified from cells lysed under non-
denaturing conditions using the linear ubiquitin chain-binding coiled-zinc finger (CoZi)
domain of NEMO (23). Upon treatment with LPS, linear ubiquitin levels dramatically
increased and peaked by 2 hours post-stimulation (Fig. 2A). By 8 hours after LPS
treatment, linear ubiquitination declined to levels comparable to unstimulated levels (Fig.
2A, Fig. S2). Interestingly, the linear ubiquitin-specific antibody, but not the CoZi domain,
pulled down linear ubiquitin chains after 4 hours of stimulation (compare Fig. 2A-B to Fig.
S2). Unlike the CoZi pulldown, the immunoprecipitation was performed under denaturing
conditions. It is possible that the linear ubiquitin chains present in cells at an earlier
timepoint are not accessible for CoZi binding, which could explain this discrepancy and
suggests that the linear ubiquitin antibody is a more accurate tool to measure linear
ubiquitination. We next examined the kinetics of LUBAC substrate modification. The linear
ubiquitination of IRAK1, a LUBAC substrate, which is modified by mixed M1/K63 ubiquitin
chains in MyD88-dependent signaling complexes (13), also peaked at 2 hours post
stimulation in A20.2J cells (Fig. 2B) as shown by the IRAK1 smear in the IP sample.
These results demonstrate similar temporal regulation of both LUBAC activity and LUBAC
substrate modification upon TLR4 activation in A20.2J B-cells.
To further test the role of HOIP ubiquitination upon TLR4 stimulation, we
complemented A20.2J cells carrying a homozygous HOIP deletion (HOIP
-/-
) (10) with
lentiviral constructs expressing either mouse Flag-tagged HOIP-WT or Flag-tagged
HOIP-K1052R (homologous to human K1056 and referred to as HOIP-KR). HOIP-WT
103
and HOIP-KR expression was equivalent to endogenous HOIP and both stabilized HOIL-
1L expression (Fig. 2C). Furthermore, both HOIP-WT and HOIP-KR had similar abilities
to associate with endogenous LUBAC components HOIL-1L and Sharpin (Fig. 2D). Next,
HOIP
+/+
and HOIP
-/-
cells complemented with HOIP-WT or vector alone were compared
in their ability to induce the linear ubiquitination of IRAK1 upon LPS treatment.
Complemented cells expressing HOIP-WT, but not cells expressing vector alone, were
able to trigger IRAK1 linear ubiquitination at equivalent levels to HOIP
+/+
cells (Fig. 2E).
This demonstrates that HOIP
-/-
cells complemented with exogenous Flag-tagged HOIP
behave similarly to HOIP
+/+
cells and exhibit stimulation-dependent LUBAC activity,
providing a system to study the function of HOIP ubiquitination.
HOIP is ubiquitinated upon TLR4 stimulation
We next used the A20.2J complement cell system to examine the ubiquitination of HOIP
in the context of immune receptor signaling. TLR4 stimulation led to the appearance of
high molecular weight HOIP species, suggesting that HOIP underwent post-translational
modification (Fig. 3A). Interestingly, these high molecular weight bands appeared after
total linear ubiquitination levels peaked (Fig. 2A). To investigate whether this modification
was ubiquitination at lysine-1056, we compared the ubiquitination status of HOIP in HOIP-
WT and HOIP-KR complement cells upon TLR4 stimulation. Indeed, HOIP-WT but not
HOIP-KR underwent ubiquitination as shown by the appearance of high molecular weight
HOIP bands as well as ubiquitinated HOIP species (Fig. 3B). Collectively these data
indicate that HOIP is ubiquitinated at lysine-1056 upon TLR4 stimulation, and that this
modification correlates with a decrease in total linear ubiquitin levels.
Mutation of HOIP lysine-1056 increases LUBAC enzymatic activity
While HOIP-WT and HOIP-KR had similar in vitro activity (Fig. S1F), we wanted to
determine if the ubiquitination of lysine-1056 altered HOIP enzymatic activity in cells. We
used conditions where LUBAC activity is stimulation-dependent (Fig. 2A) as well as a
system where HOIP is constitutively active. Transfection of HOIP and HOIL-1L in 293T
cells leads to constitutively active LUBAC enzymatic activity independent of immune
receptor simulation (5). Transfection of HOIP-K1056R, but not K640R, in 293T cells
104
resulted in the elevation of linear ubiquitination relative to that of HOIP-WT (Fig. 4A),
suggesting that ubiquitination at lysine-1056 could negatively regulate HOIP enzymatic
activity. While HOIP could be modified with K48-ubiquitin chains for protein degradation,
the proteasome inhibitor MG132 treatment did not alter linear ubiquitin levels and the
HOIP-KR mutant showed similar HOIL-1L binding affinity as HOIP-WT (Fig. 2D, Fig. S3A-
B). This showed that enhanced linear ubiquitin levels generated by HOIP-KR were
independent of protein degradation and HOIL-1L binding activity. Together, these data
suggest that ubiquitination at lysine-1056 could alter the intrinsic enzymatic activity of
HOIP.
To confirm these results in the context of immune receptor stimulation, the
complemented cell lines used in Fig. 2 were stimulated with LPS for various lengths of
time. Similar to transfection in 293T cells, stable expression of the HOIP-KR mutant in
A20.2J cells also showed elevated linear ubiquitination upon LPS stimulation relative to
that of the HOIP-WT (Fig. 4B-D). Interestingly, A20.2J HOIP-WT cells showed an
increase of intracellular linear ubiquitination upon LPS stimulation that peaked at 2 hours,
rapidly decreased at 4 hours, increased slightly at 6 hours, and then decreased at 8 hours
post-stimulation (Fig. 4C). By striking contrast, A20.2J HOIP KR cells showed a
continuous increase in linear ubiquitination upon LPS stimulation (Fig. 4C). Neither
LUBAC protein levels nor LUBAC subunit mRNA levels were altered upon LPS
stimulation in A20.2J cells (Fig. 4C, 4F, Fig. S3C), suggesting that changes in linear
ubiquitination are not due to fluctuations in LUBAC expression levels. Thus, both transient
and stable cell systems for measuring HOIP activity demonstrated the enhanced
enzymatic activity of HOIP-KR compared to HOIP-WT.
Next, we used fluorescence-activated cell sorting (FACS) to quantify intracellular
linear ubiquitination levels using a linear ubiquitin-specific antibody. A20.J2 HOIP
-/-
,
HOIP-WT and HOIP-KR cells were stimulated with LPS for various times and subjected
to intracellular anti-linear ubiquitin staining followed by FACS analysis. Concordant with
Fig. 2A, this intracellular anti-linear ubiquitin staining also showed that linear ubiquitination
peaked at 2 hours in HOIP WT cells upon LPS stimulation and declined thereafter,
whereas no increase was detected in HOIP
-/-
cells (Fig. S3D-E). Interestingly, the signal
in unstimulated HOIP
-/-
cells was lower than that in HOIP
+/+
cells, suggesting the presence
105
of low levels of linear ubiquitination in resting cells, as was seen by immunoprecipitation
(Fig. S3F, Fig. 4F). Using these observations, we defined linear ubiquitination positive
cells as those cells with a fluorescence intensity higher than in resting cells and then
compared the linear ubiquitination levels between HOIP-WT and HOIP-KR cells. This
showed that the linear ubiquitination levels were higher overall in HOIP-KR cells than in
HOIP-WT cells after 2 hours of LPS stimulation (Fig. 4E). We finally compared linear
ubiquitination levels of LUBAC substrate IRAK1, a key TLR4 signaling molecule (13). The
linear ubiquitination levels of IRAK1 were not only elevated after 2 hours of LPS
stimulation but also remained elevated after 2 hours in HOIP-KR cells compared to those
in HOIP-WT cells, (Fig. 4F). These data demonstrate that mutation of the HOIP lysine-
1056 ubiquitination site results in sustained LUBAC enzymatic activity upon TLR4
stimulation.
HOIP ubiquitination triggers HOIP conformational change
When we compared the ubiquitination status of HOIP-WT and HOIP-KR before and after
TLR4 stimulation, the HOIP-KR mutant showed far less ubiquitination than HOIP-WT but
showed similar protein stability to HOIP-WT (Fig. 3B), suggesting that ubiquitination may
not trigger HOIP degradation. To address whether the ubiquitination at the HOIP K1056
residue affects LUBAC enzymatic activity by altering its conformation, we developed the
conditions for a fluorescence resonance energy transfer (FRET) assay (24). FRET-HOIP
constructs contained an amino terminus enhanced yellow fluorescent protein (eYFP) and
carboxyl terminus enhanced cyan fluorescent protein (eCFP) fusion (Fig. 5A). The HOIP-
WT-FRET fusion construct produced linear ubiquitin chains in 293T cells (Fig. S4A), and
induced IkBa phosphorylation and degradation in A20.2J cells (Fig. S4B), indicating that
the FRET-HOIP-WT fusion protein is functionally similar to HOIP-WT in cells despite its
reduced expression compared to HOIP-WT expression in A20.2J cells (Fig. S4B-C). Both
FRET and linear ubiquitination levels were higher in unstimulated 293T cells transfected
with FRET-HOIP-KR compared to FRET-HOIP-WT, indicating that FRET-HOIP-KR also
is functionally similar in 293T cells compared to HOIP-KR (Fig. 5B). To compare FRET
signals between the HOIP constructs, a direct YFP-CFP (FRET) fusion construct was
transfected as a positive FRET control and individual YFP and CFP-HOIP fusion
106
constructs were used to correct for background FRET levels resulting from YFP emission
in the CFP spectrum and CFP emission in the YFP spectrum, respectively. The ratio of
fluorescent emission at 486nm (CFP) to 535nm (YFP) was measured in live cells. While
FRET-HOIP-KR had a FRET signal equivalent to CFP-HOIP background levels, FRET-
HOIP-WT exhibited a positive FRET signal (Fig. 5C). In order to determine whether there
were conformational changes in HOIP upon LPS stimulation, FRET-HOIP-WT and FRET-
HOIP-KR constructs were electroporated into HOIP
-/-
A20.2J cells, followed by LPS
stimulation. CFP and YFP fluorescence intensity was measured hourly following LPS
stimulation (Fig. 5D). The relative FRET intensity of the FRET-HOIP-WT construct rapidly
increased upon LPS stimulation, reached a peak level at 2 hours, and declined at 3 hours
after stimulation, suggesting that a conformational change occurs in HOIP-WT upon LPS
stimulation. In striking contrast, the FRET intensity for FRET-HOIP-KR was not altered
upon LPS stimulation. A control YFP-CFP (labeled FRET) fusion also showed no
alteration of its FRET intensity upon LPS stimulation (Fig. 5D). Collectively, this suggests
that HOIP dynamically undergoes conformational change upon LPS-induced TLR4
activation in a K1056 ubiquitination-dependent manner.
Ubiquitination of HOIP-K1056 blocks NF-κB activation
As HOIP enzymatic activity is required for efficient TLR4- and CD40-mediated NF-κB
activation in B cells (6, 7), we compared the levels of NF-κB activation between A20.2J
HOIP-WT and HOIP-KR complemented cells. Interestingly, no obvious differences in NF-
κB activation were detected between WT and KR cells during the initial response (30
minutes) to LPS treatment as measured by IκBα phosphorylation and degradation (Fig.
S5A). In contrast, HOIP-KR cells had considerably elevated levels of IκBα
phosphorylation compared to HOIP-WT cells during extended LPS treatment (60 to 480
minutes) (Fig. 6). Next, the mRNA levels for NF-κB inducible genes tnf (TNF-α), icam1
(ICAM1), nfkbia (IκBα), and tnfaip3 (A20) were determined by qRT-PCR. The mRNA
levels for NF-κB-inducible genes in HOIP-WT cells rapidly increased at 1-hour post LPS
stimulation and immediately decreased to levels comparable to the initial unstimulated
conditions (Fig. 6B). Strikingly, HOIP-KR cells showing sustained and enhanced linear
ubiquitination exhibited continuously elevated mRNA levels of NF-κB-induced genes
107
upon LPS stimulation (Fig. 6B). However, this difference occurred specifically during
TLR4 stimulation but not CD40 stimulation since comparable levels of CD40 stimulation-
induced NF-κB activation were observed in both HOIP-WT and HOIP-KR cells as
measured by IκBα phosphorylation and NF-κB-dependent gene expression (Fig. 6C-D,
Fig. S5B). We also attempted to use A20.2J cells to investigate HOIP ubiquitination in
TNF-α signaling. However, due to the lack of TNF-α receptor surface expression of
A20.2J cells (Fig. S5C-E), we were not able to examine NF-κB activation and HOIP
ubiquitination upon TNF-α signaling. Collectively, these data strongly suggest that TLR4-
induced NF-κB activation is negatively regulated by ubiquitination of the HOIP K1056
residue, while CD40-induced NF-kB activation is independent of HOIP K1056
ubiquitination.
Discussion
While ubiquitination of HOIP has been detected at multiple residues (18, 19),
detailed functions of these ubiquitinations have not been described. Here, we
demonstrate that HOIP dynamically undergoes a conformational change upon LPS-
induced TLR4 activation in a K1056 ubiquitination-dependent manner and that the
enzymatic activity of HOIP is negatively regulated through the ubiquitination of K1056.
Interestingly, HOIP was modified by multiple ubiquitin chain linkages including linear (M1),
K11, K48, and K63 linked ubiquitin chains, that were all drastically reduced in the HOIP
K1056R mutant. A previous report has demonstrated the presence of hybrid K63 and
linear ubiquitin chains in IL-1R signaling, which activates NF-κB through a mechanism
similar to TLR4 stimulation (13). This indicates that while mixed ubiquitin chains facilitate
NF-κB activation by recruiting adaptor molecules, they may also inhibit NF-κB activation
by suppressing HOIP function. Thus, mixed ubiquitin chains could have substrate-specific
functions. Furthermore, previous mass spectrometry of HOIP found ubiquitination at the
K99 residue in the region that binds linear ubiquitin deubiquitinase Otulin (25, 26),
suggesting that the K99 ubiquitination may regulate the HOIP-Otulin interaction. Thus,
HOIP itself undergoes various ubiquitinations, which ultimately regulates LUBAC activity
for intracellular signal transduction.
Our study found that ubiquitination of HOIP at K1056 alters HOIP conformation
and subsequently decreases its enzymatic activity. The K1056 residue of HOIP exhibits
108
a high degree of evolutional conservation among vertebrates (from Homo sapiens to
Danio rerio) and is located in the C-terminal LDD of HOIP that is essential for its enzymatic
activity. Our data suggest that when the K1056 residue undergoes ubiquitination, HOIP
adopts a conformational change where its amino and carboxyl termini are in close
proximity, and that this conformational change inhibits the ability of HOIP to synthesize
linear ubiquitin chains and/or modify substrates with linear ubiquitin chains. Specifically,
the Npl4 Zinc Finger (NZF) domain is a compact zinc-binding module found in many
proteins involved in ubiquitin-dependent processes, including HOIP, and has been shown
to bind K63-ubiquitin chains (27). It is thus possible that the K1056 linked ubiquitin chain
on HOIP may bind its central NZF domain, resulting in the closed conformation of the
carboxyl RBR enzymatic domain, thereby reducing its enzymatic activity (Fig. 7). In fact,
while the RBR family ligases have been shown to adopt autoinhibited conformations
during resting conditions, little is known about the mechanistic similarities during RBR
inactivation (14). While the full length structure of HOIP is not known, structural studies
suggest that RBR family member Parkin has a high degree of flexibility and that significant
rearrangement occurs during enzymatic activation (28, 29). As conformational change is
essential for enzyme activation, it is reasonable to expect that rearrangement could
subsequently impair enzymatic activity. Thus, together with the known mechanism of
Parkin RBR ligase inactivation (15), our study on HOIP suggests that post-translational
modification and subsequent conformational change may be a general mechanism to
inactivate the enzymatic activity of RBR family E3 ligases.
LUBAC has immune receptor and cell type-specific signaling functions (30).
Intriguingly, we observed a specific role for the K1056 ubiquitination of HOIP in TLR4, but
not CD40, signaling. CD40 is a member of the TNF receptor (TNFR) superfamily with
shared signaling components to the TNFR1, including cIAP1/2 that is required for LUBAC
recruitment to receptor signaling complexes (31). In contrast, TLR4 is functionally similar
to IL-1R since both require the signaling molecules MyD88, IRAK1, and IRAK4, all of
which are the known linear ubiquitination substrates of LUBAC (13). Further, IL-1R
signaling, but not TNF signaling, requires K63-linked ubiquitin chains for NF-κB activation
and LUBAC activation (13, 32). These functional differences required for LUBAC
activation between these two pathways support the idea of receptor-specific negative
109
regulation of LUBAC activity. Indeed, TLR and IL-1R have negative regulatory
mechanisms distinct from TNF signaling (33). Thus, like seen in many receptor mediated
signaling pathways, the regulation of LUBAC activity, through post-translational
modification at a specific site of HOIP, is receptor-specific. Future studies could
investigate the receptor specific regulatory roles of post-translational modification of
HOIP.
It is intriguing that during TLR4 stimulation, the difference in signal transduction
between HOIP-WT and HOIP-KR cells became evident 2 hours after stimulation, which
coincided with HOIP ubiquitination. This suggests that the KR mutation does not impact
the initial response to TLR4 activation, but rather prevents the inactivation of intracellular
signaling by permitting continued linear ubiquitination of substrates. As HOIP activity
appears to be terminated after the first round of NF-κB gene induction, it is plausible to
hypothesize that one or more NF-κB-inducible E3 ligases mediates the K1056
ubiquitination of HOIP. It will be interesting to investigate which E3 ligase targets the HOIP
K1056 specifically in TLR4 signaling. Further work on HOIP ubiquitination could
investigate how HOIP activity is regulated in different immune signaling pathways as our
work suggests the presence of multiple regulatory mechanisms. This idea is supported
by the fact that multiple HOIP isoforms have been detected in humans and specifically,
one of these lacks the Otulin-binding PUB domain, implying a need for Otulin-independent
regulation of LUBAC activity (34). However, even in this context Otulin activity may still
be required to disassemble linear ubiquitin chains after HOIP activity has been
terminated. Knowledge of the precise regulatory mechanisms of LUBAC activity could
present a strategy to therapeutically target inflammatory signaling with high specificity.
110
Materials and Methods
Cell culture, stimulation, and complement cell generation
The mouse A20.2J B-cell line has been previously described (10). Cells were cultured in
RPMI supplemented with 10% FBS, 100 U/ml penicillin, 100µg/ml streptomycin and 10µm
2-mercaptoethanol (2ME). Cells were stimulated with 10µg/ml LPS (E. coli 0127:B8;
Sigma-Aldrich) for indicated times. To induce CD40 stimulation, cells were treated with
5µg/ml of CD40L (Peprotech) for indicated times. TNF-α (Biolegend) was added to cells
at 20ng/ml. 293T cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml
penicillin and 100µg/ml streptomycin. For complemented cell generation, lentiviruses
were produced in 293T cells by calcium phosphate transfection using the pCDH-CMV-
MCS-EF1-hygro system (System Biosciences). Virus was collected two days after
transfection, filtered, and concentrated by ultracentrifugation at 20,000 RPM for 2 hours.
A20.2J cells were spin-infected at 2,000 RPM for 30 minutes in RPMI containing 8 µg/ml
polybrene and 100 µl concentrated virus. Two days after infection, media was changed
to include 200 µg/ml Hygromycin-b.
qRT-PCR
RNA was isolated from A20.2J cells with 1ml Tri Reagent (Sigma-Aldrich). DNA was
removed by treatment with DNase I (Sigma-Aldrich). RNA was reverse-transcribed to
generate cDNA using iScript (Bio-Rad Laboratories). cDNA was diluted 1:10 with water
and quantified by qRT-PCR using the iQ SYBR Green Supermix kit (Bio-Rad
Laboratories). Reactions were cycled at 95°C for 5 min, followed by 40 cycles of 95°C for
15 s and 60°C for 30 s, followed by melt curve analysis and analyzed on a CFX96 PCR
machine (Bio-Rad Laboratories). Threshold cycle (Ct) ratios were determined by
normalizing to 18S RNA and a vector complementation control sample using the following
equation: relative expression = 100*2
−(ΔΔCt)
, where ΔΔCt = (Ctgene − Ct18S) − (ΔCtVec
unstimulated). All values were normalized to the unstimulated vector condition. Primer
sequences have previously been validated (5).
111
In vitro linear ubiquitin chain formation assay
HOIL-1L-6xHIS and HOIP-K1056R-6xHIS were cloned into the pET-duet-1 vector,
expressed in BL21 DE3 RIPL bacteria (Agilent Technologies), and purified from cultures
as previously described (5). HOIP-Cterm (amino acids 699-1072), E1 (Ube1), E2
(UbcH5c), and ubiquitin were purchased from Boston Biochem. In vitro linear
ubiquitination assays contained 200 ng E1, 400 ng E2, 1 µg HOIL-1L/HOIP-6xHIS
purification prep, 20 mM Tris, pH 7.5, 5 mM DTT, 5 mM MgCl2, 2 mM MgATP (Boston
Biochem), and 10µg ubiquitin. Assays were incubated for indicated times at 37°C and
stopped by adding LDS sample buffer (Life Technologies) and heating at 70°C for 10
minutes.
Immunoprecipitation
For linear ubiquitin immunoprecipitation (IP), cells were lysed in linear ubiquitination IP
buffer (LUIP: 5M urea, 135 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 2 mM N-
ethylmaleimide, and complete protease inhibitor cocktail [Roche]). 5x10e6 cells were
used per condition. Lysates were precleared with sepharose beads for one hour at room
temperature. 0.25 µg of linear ubiquitin antibody was added (Genentech) to each sample
and incubated overnight at room temperature with end-over-end mixing. Protein A/G
beads were then incubated with samples for 2 hours at room temperature and samples
were washed with LUIP twice, then PBS twice. Samples were eluted for immunoblotting
from beads by adding LDS sample buffer supplemented with 2.5mM DTT and heating at
70°C for 10 min. For denaturing IP experiments to detect ubiquitinated HOIP species,
cells were lysed in 100µl of RIPA buffer (50mM Tris pH 7.4, 1% NP-40, 0.5% sodium
deoxycholate, 150mM NaCl) supplemented SDS to reach a final concentration of 1% SDS
to denature and disrupt protein-protein interactions. Samples were sonicated and then
diluted to 1ml in RIPA buffer. Lysates were precleared with sepharose beads for one hour
at 4°C then incubated with anti-Flag antibody (Sigma) overnight at 4°C. Protein A/G beads
were incubated with lysate/antibody mixtures for 2 h before washing 3 times in RIPA
buffer and heating at 70°C in 2× Laemmli dye for 10 min to elute protein for
immunoblotting. For precipitation of LUBAC, cells were lysed in IP buffer (1% NP-40, 50
mM Tris, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, and complete protease
112
inhibitor cocktail [Roche]) and sonicated for 20 s at 10% amplitude. Lysates were
precleared with sepharose beads for one hour at 4°C then incubated with anti-Flag
antibody (Sigma) overnight at 4°C. Protein A/G beads were incubated with
lysate/antibody mixtures for 2 h before washing 3 times in IP buffer and IP beads were
heating at 70°C in 2× Laemmli dye for 10 min to elute protein for immunoblotting. Pull
down of linear ubiquitin with CoZi-GST was performed as previously described (5). Where
293T cells were used, cesium chloride-purified plasmid DNA preparations were
transfected by PEI as previously described (35). Cell lysates were collected 24 hours after
transfection.
Immunoblotting
Cell lysates were collected in RIPA buffer and quantified by Bradford protein assay
(Thermo Fisher Scientific) to ensure an equal amount of protein loading. Proteins were
separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories) by
semi-dry transfer at 25V for 30 min. For linear ubiquitin immunoblots, SDS-PAGE was
transferred to nitrocellulose (Bio-Rad Laboratories) by wet transfer at 4°C for 2 h at 30V.
Membranes were blocked in 5% milk in PBST, or 3% BSA in TBST for pIκBα, and probed
overnight with indicated antibodies in 3% BSA, except for the linear ubiquitin antibody,
which was incubated at room temperature for 1 h. Primary antibodies included: mouse
IκBα (1:1,000; Cell Signaling Technology), mouse pIκBα (1:1,000; Cell Signaling
Technology), actin (clone C4; Santa Cruz Biotechnology, Inc.), GAPDH (1:5000;
GeneTex), p84 (1:2000; GeneTex), V5 (1:2,000; Life Technologies), Flag (1:2,000;
Sigma-Aldrich), and HA (1:2,000; Covance). Antibodies against HOIP, HOIL-1L, and
Sharpin have been previously described (11). The linear ubiquitin (M1), K11-ubiquitin,
K48-ubiquitin, and K63-ubiquitin antibodies were a gift from Genentech and have been
previously characterized in detail (22). Samples for specific ubiquitin chain immunoblots
were separated by SDS-PAGE and transferred to nitrocellulose by wet transfer at 4°C,
30 V, for 2 h. Membranes were blocked in 5% milk in PBST, probed with primary antibody
(1:2,000) for 1 h at room temperature, washed, probed with TrueBlot-HRP (1:300; Thermo
Fisher Scientific), and then developed with ECL reagent (Thermo Fisher Scientific). For
all other antibodies, appropriate HRP-conjugated secondary antibodies were incubated
113
on membranes and bands were developed with ECL reagent and imaged on an LAS-
4000 imager (Fuji). For IP with Western blots, the Clean Blot HRP secondary (1:300;
Thermo Fisher Scientific) was substituted.
Flow Cytometry
A20.2J cells (1x10
6
) in 1ml of media were treated with 10 µg/ml LPS for indicated times.
Cells were collected by centrifugation and fixed with ice-cold methanol for 15 minutes at
-20°C. After fixation, cells were washed with PBS and blocked with 3% BSA in PBST for
1 hour at room temperature. Cells were stained with a linear ubiquitin specific antibody
(Genentech) or human isotype control for 1 hour at room temperature and then washed
two times with PBS. Next cells were incubated with APC-anti-human secondary for 1
hour. Data was acquired with a BD FACSCanto II flow cytometer and analyzed with
FlowJo software
Cloning
Mouse HOIP was cloned from cDNA derived from B6 mouse bone marrow. It was PCR-
amplified to include an amino terminal Flag tag along with 5’-NheI and 3’-NotI digestion
sites and cloned into the lentiviral pCDH-CMV-MCS-EF1-hygro vector. To clone human
and mouse HOIP-FRET constructs, we generated a pIRES-Flag-eYFP-eCFP (pIRES-
FRET) construct. Enhanced yellow fluorescent protein (eYFP) was PCR-amplified with
5’-NheI and 3’-EcoRI digest sites and cloned into pIRES-Flag. Enhanced cyan fluorescent
protein (eCFP) was PCR-amplified with 5’-MluI and 3’-XbaI and cloned into both pIRES-
Flag and pIRES-Flag-eYFP. Human and mouse HOIP were separately PCR amplified
with 5’-EcoRI and 3’-MluI digest sites and cloned into the following vectors: pIRES-Flag,
pIRES-Flag-eYFP, pIRES-Flag-eCFP, and pIRES-Flag-eYFP-eCFP.
FRET Assay
For experiments in A20.2J HOIP
-/-
cells, Neon transfection (Life Technologies) was used
to electroporate HOIP-FRET constructs. The 10 µl tip was used with cells at a
concentration of 7.6x10e6 cells/ml in buffer R. 1.5 µg of plasmid DNA was added per 10µl
of cells. The cells were pulsed with 1300V for 20ms with 2 pulses. Transfection efficiency
114
was verified by counting eYFP-positive cells 24 hours post transfection as well as by
immunoblot. For FRET analysis, four 10 µl transfections were used per condition. Cells
were pelleted 24 hours post-transfection and resuspended in 100 µl FluoroBrite DMEM
(LifeTechnologies) supplemented with 10% FBS, 100 U/ml penicillin, 100µg/ml
streptomycin and 10 µm 2ME. For 293T FRET experiments, cells were transfected with
FRET-HOIP constructs. 24 hours after transfection cells were lysed with IP buffer and
lysate was used for FRET analysis. Cells and lysates were imaged with the EnVision
Multilabel Reader (Perkin Elmer) and emission readings were recorded at 486nm for CFP
and 535nm for YFP. As a negative control, cells were transfected with either HOIP-eCFP
alone or HOIP-eYFP alone. A direct eYFP-eCFP fusion FRET construct was used as a
positive control. Sample emission was measured prior to stimulation and at indicated
times after 10µg/ml LPS was added to media. To correct background in fluorescent
emission values, the emission of HOIP-eCFP in the YFP channel was subtracted from
the YFP emission value of all samples. The emission of HOIP-eYFP in the CFP channel
was subtracted from the CFP emission value of all samples. The corrected values were
used for the FRET ratio which was the value of CFP emission divided YFP emission.
Values were then normalized to the eYFP-eCFP positive FRET construct.
Mass Spectrometry
Bands corresponding to high molecular weight HOIP species were cut from an 8% SDS-
PAGE gel and sent to the Harvard Taplin mass spectrometry facility for processing and
analysis. Protein was digested with trypsin and peptides were analyzed for modification
with ubiquitination and phosphorylation.
Statistical Analysis
All experiments were performed a minimum of three times and representative data are
presented here. For experiments with statistical analysis, a two-tailed Student’s t test and
p-values of <0.05 were considered significant.
115
Acknowledgments
This work was partly supported by CA82057, CA31363, CA115284, CA180779,
DE023926, HL110609, AI073099, AI116585, Hastings Foundation, Fletcher Jones
Foundation, and GRL Program. We thank Drs. Vishva Dixit, Junying Yuan, Si-Yi Chen,
and Blossom Damania for reagents. Finally, we thank all Jung’s laboratory members for
their discussions.
116
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Figure Legends and Figures
FIG 1. Identification and characterization of HOIP ubiquitination
(A) LUBAC was purified and separated on an SDS-PAGE gel. Bands corresponding to
elevated molecular weight HOIP species were cut and protein was digested with trypsin
for mass spectrometry analysis. A search for post-translationally modified LUBAC
peptides revealed that HOIP was modified by ubiquitin at lysine-640 and lysine-1056.
While Lysine-640 is not conserved between mouse and human, Lysine-1056 is highly
conserved in vertebrates and is located in the linear ubiquitin chain determining domain
(LDD) of HOIP.
(B) Flag-tagged HOIP constructs with various lysine-to-arginine mutations were
cotransfected in human 293T cells with HA-Ubiquitin constructs. Cells were lysed under
denaturing conditions (1% SDS), followed by 10 fold dilution with lysis buffer for
renaturation. Flag-HOIP was immunoprecipitated with anti-Flag and immunoblotted with
anti-HA to detect the ubiquitinated forms of HOIP. Lysates were also used for
immunoblotting with anti-Flag (bottom panel).
(C) V5-tagged C-terminus HOIP constructs were cotransfected in human 293T cells with
HA-Ubiquitin lysine point mutant (Ubiquitin-K48R or Ubiquitin-K63R) constructs. Lysates
were immunoprecipitated with anti-V5, followed by immunoblotting with anti-HA. Lysates
were also used for immunoblotting with anti-V5 or anti-HA.
(D) V5 tagged C-terminus HOIP constructs were cotransfected in human 293T cells with
HA-Ubiquitin lysine mutant constructs where all lysines, except for the indicated residue,
were mutated to arginine: Ubiquitin-K48 or Ubiquitin-K63 can only form K48 or K63 linked
ubiquitin chains, respectively. Experiment was performed as in B.
(E) V5 tagged C-terminus HOIP constructs were cotranfected in human 293T cells with
HA-Ubiquitin constructs. Immunoprecipitation and immunoblotting were conducted as in
B. Samples were probed with ubiquitin linkage-specific antibodies to determine ubiquitin
chain linkages modifying HOIP.
Data are representative of three independent experiments.
121
FIG 2. Kinetics of LUBAC activity and complementation of HOIP deficient cells
(A) Mouse A20.2J B-cells were stimulated with 10µg/ml LPS to activate TLR4 signaling
and induce LUBAC enzymatic activity. Cellular linear ubiquitin levels were determined by
immunoprecipitation with a linear ubiquitin specific antibody. Resting cells have low levels
of linear ubiquitin relative to cells stimulated with LPS for 1 hour.
(B) Performed as in A, with earlier time points as well as immunoblot for IRAK1.
(C) Mouse A20.2J HOIP
-/-
cells were complemented with vector, HOIP-WT, or HOIP-KR
by lentiviral infection. LUBAC expression in complement cells was compared to A20.2J
HOIP
+/+
cells by immunoblot analysis.
(D) HOIP-Flag was immunoprecipitated from lysates of cell lines generated in A using
Flag or IgG control antibody to determine LUBAC formation.
(E) To verify function of complement cell lines, cells were stimulated with 10µg/ml LPS
for 1 hour and then lysed in LUIP buffer. A linear ubiquitin-specific antibody was used to
precipitate linear ubiquitin chains, followed by immunoblotting with anti-IRAK1.
Data are representative of three independent experiments.
FIG 3. HOIP is ubiquitinated upon TLR4 stimulation
(A) A20.2J mouse B-cells were stimulated with LPS and lysed under denaturing
conditions. Endogenous HOIP was immunoprecipitated, separated by SDS-PAGE and
analyzed by immunoblot. High molecular weighted HOIP bands (>120 KDa) appeared
after LPS stimulation.
(B) Complemented mouse A20.2J HOIP
-/-
cells shown in Fig. 2C were stimulated with
LPS for indicated times and lysed under denaturing conditions. Anti-IgG (control) or anti-
Flag (HOIP) immunoprecipitated samples were prepared as in A. Long exposure of Flag
immunoblot revealed the presence of high-molecular weighted HOIP bands.
Data are representative of three independent experiments.
FIG 4. Mutation of HOIP lysine-1056 increases LUBAC enzymatic activity
(A) HOIP constructs were transfected into human 293T cells in the presence or absence
of HOIL-1L. At 24 hours after transfection cells were lysed and LUBAC protein expression
and linear ubiquitin chain levels were determined by immunoblot analysis of cell lysates.
122
(B) A20.2J HOIP
-/-
complemented cells from Fig. 2C were stimulated with 10µg/ml LPS to
activate TLR4 signaling and induce LUBAC enzymatic activity. Endogenous linear
ubiquitin levels were determined by immunoprecipitation with a linear ubiquitin specific
antibody.
(C) Performed as in B with LPS stimulation for varying time points to compare linear
ubiquitin levels between WT and KR cells.
(D) Densitometry analysis of linear ubiquitin immunoblot from C. All values are relative to
WT cells prior to stimulation.
(E) A20.2J HOIP
-/-
complemented cells stimulated as in C were stained for linear ubiquitin
using M1Ub-APC and linear ubiquitin positive cells were quantified by flow cytometry.
(F) A20.2J HOIP
-/-
complemented cells were stimulated as in C. Immunblot for IRAK1
shows the presence of linear ubiquitin chains on IRAK1 upon TLR4 activation.
Data are representative of three independent experiments.
FIG 5. TLR4 stimulation induces HOIP conformational change
(A) Diagram of HOIP-FRET constructs with amino terminal YFP fusions and carboxyl
terminal CFP fusions.
(B) Function of HOIP-FRET constructs as determined by immunoblot for linear ubiquitins
in 293T cell lysates containing either HOIP-WT or HOIP-FRET.
(C) HOIP-FRET constructs were transfected into 293T cells. At 24 hours after
transfection, cells were lysed and FRET was measured. FRET values were calculated by
subtracting background fluorescence and dividing fluorescence at YFP by fluorescence
at CFP. Data are representative of three independent experiments.
(D) HOIP-FRET constructs were transfected using the Neon system (Life Technologies)
into A20.2J HOIP
-/-
cells. At 24 hours after transfection, cells were resuspended in
FluoroBrite media, and fluorescence was measured using an Envision plate reader. After
addition of LPS, fluorescence readings were taken at indicated times. FRET was
calculated as described in Materials and Methods, Relative FRET = FRET (X minutes) /
FRET (0 minutes). HOIP-eCFP and HOIP-eYFP constructs were used as negative
controls.
Data are representative of three independent experiments.
123
FIG 6. TLR4 stimulation leads to higher NF-κB activation in HOIP-KR cells than in
HOIP-WT cells
(A) A20.2J cells were stimulated with 10μg/ml LPS and lysed at intervals up to 480
minutes after treatment with LPS.
(B) Quantitative PCR using cDNA reverse transcribed from cells stimulated as in A for
indicated times. Values were first calculated relative to 18S, then were normalized to
vector cells prior to stimulation.
(C) A20.2J cells were stimulated with 10μg/ml CD40L and lysates were analyzed up to
240 minutes post stimulation.
(D) Quantitative PCR using cDNA reverse transcribed from A20.2J cells stimulated as in
C for indicated times. Values were first calculated relative to 18S, then were normalized
to vector cells prior to stimulation.
Data are representative of three independent experiments.
FIG 7. Model of HOIP structural alteration upon TLR4 stimulation
TLR4 stimulation activates HOIP enzymatic activity. The synthesis of linear ubiquitin
chains by HOIP promotes NF-κB activation. This signal however must be terminated to
prevent sustained signaling. Ubiquitination of HOIP at lysine-1056 is triggered by TLR4
stimulation and alters HOIP conformation. The change in conformation inactivates HOIP
enzymatic activity, which in turn removes a signal required for NF-κB activation, thus
facilitating termination of the immune response.
124
SUPPLEMENTAL FIGURE LEGENDS
FIG S1. The K1056 plays an important role in HOIP ubiquitination, but not in HOIP
enzymatic activity
(A) Previously published HOIP crystal structure of the enzymatically active carboxyl terminus of
HOIP in complex with ubiquitin. The structure reveals that lysine-1056 is on an exposed region of
the active enzyme (as indicated by the arrow), suggesting it is accessible for modification by
another E3 ligase.
(B) Regions of human HOIP included in carboxyl terminus constructs.
(C) HA tagged (which contains no lysine residues) HOIP constructs containing the RBR and LDD
domains were expressed in 293T cells along with Flag-Ubiquitin. WT lanes contain HOIP-RBR-
LDD-WT and KR lanes contain HOIP-RBR-LDD-K1056R. Immunopreciptation was conducted
under denaturing conditions. Mutation of lysine-1056 to arginine dramatically reduced the
presence of ubiquitinated HOIP species. Data are representative of three independent
experiments.
(D) Human recombinant HOIP (amino acids 699-1072) was incubated with ubiquitin, ATP, E1,
and E2 enzymes for 2 hours. Reaction was stopped with the addition of sample buffer and
samples were analyzed by immunoblot analysis.
(E) Additional blots from experiment in Fig. 1D showing HA-Ubiquitin and K11-Ubiquitin.
(F) HOIP was co-purified with HOIL-1L from BL21 DE3 RIPL bacterial cells. Purified LUBAC was
incubated with E1, E2, and ATP for indicated times. Equal protein amounts of LUBAC containing
either HOIP-WT or HOIP-KR were used for the reactions. Reactions were incubated for indicated
times.
FIG S2. Purification of linear ubiquitination by the CoZi domain of NEMO
Mouse A20.2J B-cells were stimulated with 10µg/ml LPS to activate TLR4 signaling and induce
LUBAC enzymatic activity. The ubiquitin-binding coiled-zinc finger (CoZi) domain of NEMO fused
to GST was purified from bacteria and used to pull down linear ubiquitin from cell lysate. Data are
representative of three independent experiments.
FIG S3. Increase of intracellular linear ubiquitination by HOIP KR
(A) Flag-HOIP was expressed with or without HOIL-1L-HA in 293T cells. The proteasome inhibitor
MG-132 was added to indicated samples for 4 hours at 20µM prior to cell lysis and immunoblot
analysis.
125
(B) HOIP and HOIL-1L constructs were transfected into 293T cells and cells were lysed with NP-
40 buffer 24 hours post-transfection. Immunoprecipitation was performed separately for HOIL-1L
using HA antibody and HOIP using Flag antibody. Data are representative of three independent
experiments.
(C) Quantitative PCR using cDNA reverse transcribed from A20.2J B-cell mRNA isolated from
cells stimulated with LPS for indicated times. Samples were normalized to ribosomal 18S RNA.
Each data point represents three biological and three analytical replicates.
(D) 1x10
6
A20.2J cells were stimulated with 10µg/ml LPS for indicated times, fixed, and then
stained for linear ubiquitin (M1Ub-APC). Cells were analyzed by flow cytometry.
(E) Performed as in D. HOIP
+/+
and HOIP
-/-
cells were stimulated with LPS and stained for linear
ubiquitin (M1Ub-APC). Linear ubiquitin staining was compared between cells at indicated times.
(F) Cells stimulated as in E for direct comparison between isotype staining and linear ubiquitin
staining in HOIP
+/+
and HOIP
-/-
cells.
Data are representative of three independent experiments.
FIG S4. HOIP ubiquitination and HOIP-FRET constructs
(A) Various HOIP fusion constructs were expressed in 293T cells. Linear ubiquitin levels were
determined using immunoblot analysis.
(B) HOIP-FRET constructs were transfected using the Neon system (Life Technologies) into
A20.2J HOIP
-/-
cells. 24 hours after transfection cells were stimulated with LPS. Cells were lysed
and analyzed by immunoblot. Arrows indicate FRET-HOIP and HOIP-WT proteins.
(C) Quantitative PCR using cDNA reverse transcribed from cells prepared as in C.
FIG S5. Immunoblot analysis upon LPS or CD40L stimulation
(A) Various A20.2J cell lines were stimulated with 10μg/ml of LPS and cell lysates were subjected
to immunoblot at intervals up to 30 minutes after stimulation.
(B) Indicated cells were stimulated with 10μg/ml CD40L for up to 30 minutes and cell lysates were
subjected to immunoblot.
(C) HOIP
+/+
and HOIP
-/-
cells were stimulated with TNF-α (20ng/ml) and cell lysates were
subjected to immunoblot at intervals up to 8 hours after stimulation.
(D) Quantitative PCR using cDNA reverse transcribed from cells stimulated as in C
(E) 1x10
6
A20.2J cells were stained for cell surface receptors using CD40-PE, TNFAR-PE, or
isotype control-PE. Cells were analyzed by flow cytometry. CD40 staining served as a positive
control.
126
FIG 1
A
C
Mass Spectrometry Peptide:
R.LLQK#LTEEVPLGQSIPR.R
Mouse EDLPAYQARLLQKLREEVPLGQSI 1061
Rat EDLPAYQARLLQKLREEVPLGQSI 1047
Human EDPPAYQARLLQKLTEEVPLGQSI 1067
Chimpanzee EDPPAYQARLLQKLTEEVPLGQSI 1067
Rhesus EDPPAYQARLLQKLTEEVPLGQSI 1067
Wolf EDAPAYHARLLQKLMEEVPLGQSI 1071
Zebrafish EDEQTHFSRLLKKLMDDVPLGDKV 319
** :: *** ** .:****..:
NZF UBA RBR
K1056
LDD PUB
Flag-HOIP
HA-Ub
IP-Flag WCL
Flag-HOIP:
-
WT
K330R
K640R
K1056R
K640,1056R
CS
-
WT
K330R
K640R
K1056R
K640,1056R
CS
B
Linear-Ub
K48-Ub
K63-Ub
V5-HOIP
HOIP-Ub n
V5-HOIP
HA-Ub
V5-HOIP
HA-Ub
D
E
IP-V5-HOIP WCL
HA-Ubiquitin
WT
K63R
K48R
WT
K63R
K48R
V5-HOIP-Cterm + + + + + + + +
IP-V5-HOIP WCL
HA-Ubiquitin
WT
WT
K63
K48
WT
WT
K63
K48
V5-HOIP-Cterm + + + + + + + +
IP-V5-
HOIP WCL
Vector
HOIP-KR
HOIP-WT
Vector
HOIP-KR
HOIP-WT
HA-Ubiquitin - + + - + +
127
FIG 2
LPS (m):
0
10
30
60
120
240
Linear
Ubiquitin
IRAK1
IRAK1
WCL IP Linear Ubiquitin
LPS (h): 0 1 2 4 6 8
Linear
Ubiquitin
HOIP
Actin
IP Linear Ubiquitin WCL
Sharpin
HOIL-1L
IP-V5
IP-
Flag WCL
HOIP-WT
HOIP-KR
HOIP-WT
HOIP-KR
HOIP-WT
HOIP-KR
Rescue:
Vector
HOIP-WT
HOIP-KR
HOIP:
+/+
-/-
-/-
-/-
-/-
HOIP
HOIL-1L
Sharpin
Actin
HOIP
HOIL-1L
Sharpin
P84
IRAK1
IRAK1
IP Linear
Ubiquitin WCL
HOIP: +/+ -/-
Rescue: - Vec WT
LPS 1h: - + - + - +
A
B
C
D
E
128
LPS (h): 0 1 2 3
HOIP
IP HOIP
150
100
250
150
100
250
HOIP
(Long
Exposure)
A
IgG
IP-Flag IgG
IP-Flag WCL
HOIP-WT HOIP-KR HOIP-WT HOIP-KR
LPS (m):
0
0
30
60
120
0
0
30
60
120
0
0
30
60
120
0
0
30
60
120
Ubiquitin
Flag-HOIP
Flag-HOIP
(Long
Exposure)
HOIP-Ub
HOIP-Ub
FIG 3
B
129
HOIP-WT
HOIP-K640R
HOIP-K1056R
HOIP-WT
HOIP-K640R
HOIP-K1056R
HOIL-1L - - - + + +
Linear
Ubiquitin
HA-HOIL-1L
Flag-HOIP
293T
Actin
Vec
KR
WT
HOIP: -/- -/- -/-
LPS: - + - + - +
Linear
Ubiquitin
IP Linear
Ubiquitin
HOIL-1L
HOIP
P84
WCL
Sharpin
A20
Vector HOIP-KR HOIP-WT
LPS (h): 0 2 4 0 1 2 3 4 0 1 2 3 4
Linear
Ubiquitin
IRAK1
HOIL-1L
Sharpin
IRAK1
P84
IP Linear Ubiquitin WCL
A20
HOIP-WT HOIP-KR
LPS (h): 0 1 2 4 6 8 0 1 2 4 6 8
Linear
Ubiquitin
HOIP
Actin
IP Linear
Ubiquitin WCL
A20
FIG 4
A
B
C
F
0
8
16
24
HOIP-WT HOIP-KR
Relative to WT 0h (a.u.)
Linear Ubiquitin Levels
0h +LPS 1h +LPS 2h +LPS
4h +LPS 6h +LPS 8h +LPS
D
E
0
7
14
21
2 4
M1-Ub Positive (%)
LPS (h)
LPS Induced LUBAC Activity
Vector HOIP-WT HOIP-KR
130
FIG 5
A B
YFP
CFP RBR LDD
UBA NZF PUB
FRET
≈10-100Å
YFP
CFP
RBR
LDD
UBA NZF PUB
>100Å
NO FRET
Vector
HOIP-WT
HOIP-KR
FRET-HOIP-WT
FRET-HOIP-KR
HA-HOIL-1L + + + + +
C D
LinUb
HOIP
GAPDH
HA-HOIL-1L
293T
0
1.5
3
4.5
CFP
YFP
pFRET
HOIP-WT
HOIP-KR
HOIP-WT
HOIP-KR
- + HOIL-1L
CFP 486/YFP 535
0
1
2
3
0 80 160 240
Relative FRET Intensity
Minutes + LPS
FRET
HOIP-WT-FRET
HOIP-KR-FRET
131
FIG 6
pIκBα
IκBα
P84
Vector HOIP-WT
HOIP: -/- -/-
LPS (m):
0
60
120
240
360
480
0
60
120
240
360
480
HOIP-WT HOIP-KR
-/- -/-
0
60
120
240
360
480
0
60
120
240
360
480
A
Vector HOIP-WT HOIP-KR
B
pIκBα
IκBα
P84
Vector HOIP-WT
HOIP: -/- -/-
CD40L(m):
0
30
60
120
180
240
0
30
60
120
180
240
HOIP-WT HOIP-KR
-/- -/-
0
30
60
120
180
240
0
30
60
120
180
240
C
D
0
10
20
30
40
0 2 4
CD40L (h)
IκBα
0
4
8
12
16
0 2 4
CD40L (h)
A20
0
3
6
9
12
0 2 4
CD40L (h)
TNFα
0
3
6
9
12
0 2 4
CD40L (h)
ICAM1
mRNA Relative to Vector mRNA Relative to Vector
0
20
40
60
80
0 4 8
LPS (h)
TNFα
0
10
20
30
40
0 4 8
LPS (h)
ICAM1
0
40
80
120
160
0 4 8
LPS (h)
IκBα
0
40
80
120
160
0 4 8
LPS (h)
A20
mRNA Relative to Vector mRNA Relative to Vector
Vector HOIP-WT HOIP-KR
132
RBR LDD
UBA
NZF
PUB
K1056
M1
Ub
RBR
LDD
UBA
NZF
PUB
K1056
Ub
NF-κB
Active HOIP
Inactive HOIP
(Ubiquitinated at K1056)
FIG 7
133
FIG S1
B
Ring1 IBR Ring2
LDD
HOIP Cterm-WT
Ub
K1056
Ring1 IBR Ring2
LDD
HOIP Cterm-KR
K1056R
C
IP HA WCL
Flag-Ub + + + + + +
HA-HOIP-C
WT
KR
WT
KR
WT
KR
WT
KR
Flag-
Ubiquitin
HA-HOIP
Linear
Ubiquitin
In Vitro
HOIP-WT
HOIP-KR
HOIP-WT
HOIP-KR
Minutes: 0 0 60 60
D
E1/E2 + +
ATP + +
Ubiquitin + + +
HOIP-C + + +
E
HOIP
Ubiquitin
25
37
50
75
In Vitro
A
K11-Ub
HA-Ub
IP-V5-
HOIP WCL
Vector
HOIP-KR
HOIP-WT
Vector
HOIP-KR
HOIP-WT
HA-Ubiquitin - + + - + +
F
134
LPS (m):
0
10
30
60
240
480
Linear
Ubiquitin
IRAK1
CoZi PD
FIG S2
135
FIG S3
A
HOIP-WT
HOIP-KR
HOIP-WT
HOIP-KR
HOIP-WT
HOIP-KR
HOIP-WT
HOIP-KR
HOIL-1L-HA - - + + - - + +
MG132 - - - - + + + +
Linear
Ubiquitin
HA-HOIL-1L
Flag-HOIP
Actin
293T
C B
WCL IP Flag IP HA
HOIP-Flag
WT
KR
WT
KR
WT
KR
WT
KR
WT
KR
WT
KR
HOIL-1L-HA - - + + - - + + - - + +
HA-HOIL-1L
Flag-HOIP
293T
0
0.5
1
1.5
2
HOIP Sharpin HOIL-1L
Relative to 18s
Normalized to 0h
LUBAC mRNA
0h 1h +LPS
2h +LPS 4h +LPS
0h 0.5h 0h 1h 0h 2h 0h 3h 0h 4h
HOIP-WT
M1Ub-APC
WT 0h KO 0h
Isotype
WT 2h KO 2h
Isotype
M1Ub-
APC
0h 4h 0h 2h 0h 1h
HOIP+/+ HOIP-/- M1Ub-
APC
D E F
136
FIG S4
FRET-
HOIP-WT HOIP-WT
LPS (h): 0 1 2 0 1 2
HOIP
HOIP
YFP-HOIP
HOIP-CFP
FRET-HOIP
HOIL-1L: + + + +
Flag-HOIP
HA-HOIL-1L
Linear
Ubiquitin
293T
Flag-HOIP
IκBα
pIκBα
P84
A20
0
2
4
6
0 1 2
Fold Induction
Hours LPS
TNFα mRNA
0
2
4
6
0 1 2
Fold Induction
Hours LPS
ICAM1 mRNA
HOIP-WT
FRET-HOIP-WT
A
B
C
FRET-HOIP
HOIP-WT
137
FIG S5
pIκBα
IκBα
P84
pIκBα
IκBα
P84
A20.2J Vector
HOIP: +/+ -/-
CD40L(m):
0
5
10
15
30
0
5
10
15
30
HOIP-WT HOIP-KR
-/- -/-
0
5
10
15
30
0
5
10
15
30
A
B
0
1
2
0 4 8
TNF-ɑ (h)
IκBα
A20.2J HOIP+/+
A20.2J HOIP-/-
0
1
2
0 4 8
TNF-ɑ (h)
A20
A20.2J HOIP+/+
A20.2J HOIP-/-
C
mRNA Relative to Vector
D
CD40
Isotype
TNFAR
Isotype
HOIP +/+ HOIP -/-
TNF-α (h): 0 1 2 4 8 0 1 2 4 8
HOIP
pIkBa
IkBa
P84
Vector HOIP-WT
HOIP: -/- -/-
LPS(m):
0
5
10
15
30
0
5
10
15
30
HOIP-WT HOIP-KR
-/- -/-
0
5
10
15
30
0
5
10
15
30
E
Abstract (if available)
Abstract
The leading cause of cancer death in Africa is Kaposi’s sarcoma which is caused by infection with Kaposi’s sarcoma associated herpesvirus. Despite being a pressing human health problem, little has been done thus far to develop specific therapeutics against this infection and its associated diseases. Interestingly, during infection this virus expresses a viral homolog of a cellular G-protein coupled receptor (GPCR). The clinical relevance of GPCRs is underscored by the fact that they are the targets of 30% of all FDA approved drugs. The Kaposi’s sarcoma viral GPCR (vGPCR) is essential for virus-mediated oncogenesis: its expression is sufficient to transform cells in vitro and induce tumor formation in vivo. The fact that vGPCR plays a key role in KSHV pathogenesis and that it is a member of a gene family frequently targeted by FDA-approved drugs makes vGPCR a promising target for therapeutic intervention. In order to develop novel therapeutic agents targeting vGPCR, we engineered a thermalstable vGPCR protein to enable large scale purification of pure and homogenous receptor from mammalian cell membranes. We used this protein to identify a camelid heavy chain only “nanobody” which could specifically recognize the extracellular portion of the receptor and be used therapeutically to block vGPCR mediated oncogenesis. The purified receptor was screened against a synthetic yeast nanobody display library and we identified three unique nanobodies which bind vGPCR. We validated the binding of these nanobodies to vGPCR expressed on the cell surface and are investigating their ability to inhibit its function.
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Asset Metadata
Creator
Bowman, James Wayne, III
(author)
Core Title
Antibody discovery and structural studies of the viral oncogene ORF74
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
07/03/2019
Defense Date
05/16/2019
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
cancer, antibody, protein purification, virology, protein engineering,OAI-PMH Harvest
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application/pdf
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Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Liang, Chengyu (
committee chair
), Huang, I-Chueh (
committee member
), Jung, Jae (
committee member
)
Creator Email
jtbowmaniii@gmail.com,jwbowman@usc.edu
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https://doi.org/10.25549/usctheses-c89-180500
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cancer, antibody, protein purification, virology, protein engineering