From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0445
Received for publication, October 19, 2000, and in revised form, February 20, 2001
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ABSTRACT |
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The human interleukin (IL)-18-binding
protein (hIL-18BP) is a naturally occurring antagonist of IL-18, a
proinflammatory cytokine that is related to IL-1 Interleukin (IL)1-18 is
a recently cloned proinflammatory cytokine that was formerly known as
the interferon- The IL-18-binding protein (IL-18BP), a naturally occurring antagonist
of IL-18 which is found in both humans and mice, prevents IL-18 from
binding to its receptor (15, 16). The affinity between IL-18 and
IL-18BP is extremely high with a subnanomolar Kd
(17, 18). Functional homologs of IL-18BP have been found in poxviruses,
namely molluscum contagiosum virus, vaccinia virus, cowpox virus, and
ectromelia virus (17, 19-22), but so far not in other viruses.
Approximately 60% of the mature human IL-18BP (hIL-18BP) resembles an
immunoglobulin (Ig) domain that includes a highly conserved pair of
cysteines and a tryptophan residue. The predicted Ig domain of hIL-18BP
has about 25% amino acid sequence identity with the third Ig domain of
the type I IL-1 receptor (IL-1R1), whose x-ray structure was solved in
complex with IL-1 Construction of the Fusion Vector--
For other
purposes, we had appended a DNA segment encoding the biotinylation site
and a six-histidine tag to the 3'-end of the molluscum contagiosum
virus MC51L open reading frame. This was accomplished by polymerase
chain reaction (PCR) using DNA containing the MC51L open reading frame
as a template (19) and the primers 5'-GCCACCATGAAGGAAAGTCTTTCTCGG-3'
and
5'-GCTAGTGATGATGGTGATGGTGTTCGTGCCATTCGATTTTCTGAGCTTCGAAGATGTCGTTCAGACCCCACAGGTTTCTCAACAT-3'. The PCR product was ligated to the pTarget TA cloning vector (Promega) to form pYX26. To recover the DNA containing the biotinylation site and
six-histidine tag and to add a flexible linker GSGGGGS in front of the
biotinylation site GLNDIFEAQKIEWHE, another PCR was made using pYX26 as
the template with primers
5'-ATTAGGATCCGGTGGAGGAGGTTCTGGTCTGAACGACATCTTC-3' and
5'-CGGGGTACCCTAGTGATGATGGTGATGG-3'. The PCR product
5'-ATTAGGATCCGGTGGAGGAGGTTCTGGTCTGAACGACATCTTCGAAGCTCAGAAAATCGAATGGCACGAACACCATCACCATCATCACTAGGGTACCCCG-3', with the BamHI and KpnI sites in italics, was
digested with BamHI and KpnI and inserted into
the multiple cloning site of pcDNA3.1 (Invitrogen) to form pYX45
(see Fig. 2A). The KpnI site in pYX45 was removed
using the exonuclease activity of T4 DNA polymerase to generate
pYX45-NK.
Site-directed Mutagenesis--
The hIL-18BP open reading frame
(19) was amplified by PCR with a 5'-primer
(5'-CTATCAGCTAGCATGACCATGAGACACAACTG-3') containing an
NheI site and a 3'-primer
(5'-TAATGGATCCACCACCTCCACCCTGCTGCTGTGGACTGC-3') including an
additional GGGGS coding sequence. The last two codons of GGGGS
contained a BamHI site enabling the hIL-18BP and tag coding
sequence to be fused in-frame in pYX45-NK by using the NheI
and BamHI sites. As a result, the linker sequence between hIL-18BP and the biotinylation site and polyhistidine tag was GGGGSGGGGS. This plasmid was named pYX50-NK.
A derivative of pYX50-NK was constructed by changing the serine at
residue 103 to threonine, which created a KpnI site with the
codons of residues 102 and 103 (GT) (see Fig. 2B,
top). This unique KpnI site, together with either
the 5'-NheI site or 3'-BamHI site, allowed the
cloning of PCR products containing substitutions at residues 93, 95, 97, 99, 104, 106, 107, 108, 112, and 114 (see Fig. 2B,
top). Another derivative of pYX50-NK was constructed by
introducing a silent mutation at residue 126, which created a
KpnI site (see Fig. 2B, middle). This
unique KpnI site, together with either the
5'-NheI site or the 3'-BamHI site, allowed the cloning of PCR products containing substitutions at residue 119, 121, 130, 151, and 153 (see Fig. 2B, middle). An
additional derivative of pYX50-NK was constructed by removing the
coding sequence from residue 103 to residue 125 and creating a
KpnI site with the codons of 125 and 126 (see Fig.
2B, bottom). Residue 130 of this construct was
replaced by alanine by subcloning a fragment containing the K130A
mutation into the construct. The unique KpnI site allowed the cloning of PCR products containing multiple additional
substitutions at positions 106, 107, 114, 119, and 121 (see Fig.
2B, bottom). For each mutant, the entire hIL-18BP
coding sequence of both strands was determined to confirm that the
mutations were correct.
Protein Purification and in Vitro Biotinylation--
293T cells
in one six-well plate were transfected with 2 µg of plasmid/well
using LipofectAMINE (Life Technologies, Inc.) following the
manufacturer's protocol. After overnight incubation, the medium of
each well was replaced with 1.2 ml of serum-free Opti-MEM (Life
Technologies, Inc.). After another 3 days of incubation, the medium was
harvested, and any suspended cells were removed by centrifugation. The
supernatant was adjusted to contain 0.1% Triton X-100 and 15 mM imidazole in a total volume of 6.12 ml and was incubated
overnight at 4 °C with 0.5 ml of Ni-nitrilotriacetic acid resin
(Qiagen). The resin was then washed once with 10 ml of 15 mM imidazole in phosphate-buffered saline and twice
with 10 ml of 10 mM Tris-HCl, pH 8.0. The resin was
resuspended in 1 ml of 10 mM Tris-HCl, pH 8.0, and 125 µl
of 10 Biomix A (Avidity, Denver, Colorado), 125 µl of 10 × Biomix B (Avidity) and 3 µg of Escherichia coli biotin
holoenzyme synthetase (Avidity) were added. The resin was
incubated with shaking at 37 °C for 1-4 h, packed into a 0.8 × 4-cm disposable column (Bio-Rad), and washed with 30 ml of 15 mM imidazole in phosphate-buffered saline containing 150 mM NaCl. The recombinant protein was eluted with 2 ml of
300 mM imidazole in phosphate-buffered saline by gravity
flow at room temperature. Six 250-µl fractions were collected
manually in Eppendorf tubes. Fractions containing the biotinylated
hIL-18BP were used for analysis on a BIAcore 2000 sensor (BIAcore,
Piscataway, NJ).
Measurement of Protein Biotinylation--
Biotinylated and
control IL-18BPs were incubated with streptavidin-agarose (Pierce) or
control protein A-agarose (Pierce) for ~2 h in the presence of 0.1%
SDS and 0.1% Triton X-100. Proteins that bound to the streptavidin- or
protein A-agarose were removed by centrifugation. The amounts of
hIL-18BP remaining in the supernatants were determined by binding to
recombinant murine IL-18 (rmIL-18) (PeptroTech, Rocky Hill, NJ) that
was immobilized on a BIAcore sensor CM5 chip. The initial binding
rates, which are proportional to the IL-18BP concentrations in the
supernatants, were calculated as the average binding rates for the
first 3 min, during which time less than 2% of the IL-18BP binding
sites on the chip were occupied. The percentage of IL-18BP that was
biotinylated was determined from the initial binding rate of IL-18BP in
the supernatant after incubation with protein A-agarose minus the
initial binding rate of IL-18BP in the supernatant after incubation
with streptavidin-agarose divided by the initial binding rate of
IL-18BP in the supernatant after incubation with protein A-agarose.
Surface Plasmon Resonance--
Biotinylated hIL-18BP was
captured onto a BIAcore SA chip via the strong and virtually
irreversible interaction of biotin with streptavidin. The chip was then
washed several times with injections of 10 mM glycine, pH
1.5, to remove any loosely bound materials. Less than 300 response
units (RU) of protein was captured to prevent any mass transport effect
during the kinetic analysis. The maximal responses for IL-18 binding
(Rmax) were usually around 100 RU, which is
close to the theoretical Rmax calculated with the assumption that all captured hIL-18BP proteins are active. The
binding kinetics of hIL-18BP with recombinant human IL-18 (rhIL-18;
PeptroTech) and rmIL-18 were determined as described previously
(17).
Molecular Modeling of Mutated hIL-18BP--
The Homology and
Builder modules in the Insight II sets of programs (Molecular
Simulations Inc.) were used for model construction. The coordinates of
a published hIL-18BP structure model (18) were obtained from the
authors, and the entire structural model was optimized through energy
minimization with the Builder module. The structure model of mutated
hIL-18BP was constructed by replacing the mutated side chain with that
of alanine in the Homology module, and then the entire structure was
optimized through energy minimization with the Builder module. The root
mean square deviations of mutated and wild type hIL-18BP
structure models were determined by automatic structure alignment with
the Homology module.
Mutagenesis of hIL-18BP--
A model based on the structure of
IL-1R1 complexed to IL-1
To avoid global alterations in protein structure, in each case the
selected amino acid was changed to alanine. Additional mutations were
made at residues 114 and 130, the only two predicted by the structural
model to participate in electrostatic interactions: one substitution
maintained the same kind of charge, another changed it to the opposite
charge, and the third changed it to a polar residue. Mutations at
residue 93, 95, 97, 99, 104, 106, 107, 108, 112, and 114 also contained
a conservative threonine to serine substitution at residue 103 which
was introduced to facilitate PCR mutagenesis.
Expression and in Vitro Biotinylation of hIL-18BPs--
Because of
the high affinity between hIL-18BP and hIL-18 and the predicted minimal
structural perturbation of alanine substitutions, the sensitive and
quantitative method of surface plasmon resonance was used to determine
binding constants. The hIL-18BPs were expressed by mammalian cells as
secreted, glycosylated, C-terminal fusion proteins with a flexible
linker, followed by a 15-amino acid biotin holoenzyme synthetase
biotinylation recognition site and a six-histidine tag (Fig.
2A). The secreted hIL-18BPs
were captured on Ni-nitrilotriacetic acid resin via the six-histidine
tag. While on the resin, the proteins were biotinylated at a specific
lysine by biotin holoenzyme synthetase (24) and then washed and eluted
with imidazole. The only biotinylated protein detected after this
purification step ran as a broad 50-kDa band on SDS-polyacrylamide gel
electrophoresis (Fig. 2C), consistent with the size and
glycosylation state of hIL-18BP. This procedure had a number of
advantages over previous methods because no dialysis step was necessary
before the biotinylation reaction, and excess biotin, which could
interfere with binding to the streptavidin chip, was removed by washing
prior to elution. Approximately 90% of the protein was biotinylated by
this procedure.
Surface Plasmon Resonance Analysis of IL-18 Binding to Mutated
hIL-18BPs--
hIL-18BPs were captured individually on streptavidin
chips, and the real time association and dissociation of rhIL-18 and rmIL-18 were monitored in a BIAcore 2000 sensor. Sensorgrams for all
proteins with single amino acid substitutions were obtained by
injecting 14 nM rhIL-18, and the responses were normalized to a maximum of 100 RU. As reported previously (17), the binding of
rhIL-18 to unmutated hIL-18BP was characterized by a rapid association
phase (which is affected by both on- and off-rates) and an extremely
small decrease in response during the dissociation phase (which is only
affected by the off-rate) (Fig.
3A). The association phases of
the sensorgrams of all of the mutated proteins with single
substitutions were similar to each other (Fig. 3, A-E). By
contrast, the dissociation phases of several of the mutated proteins
differed from unmutated hIL-18BP. Alanine substitutions of some of the
selected individual residues on the predicted
Of the seven residues that were shown to affect binding, only two (106 and 114) had charged side chains, whereas the rest had hydrophobic side
chains. The effects of mutations at residues 106 and 114 on IL-18
binding were relatively small compared with mutations at 97 and 104. When the charged residues 107, 119, 121, or 130 were mutated, there was
little effect on binding even though residue 130 was predicted to
interact with hIL-18 (18). In an effort to assess whether the charged
amino acids as a group might contribute to IL-18 binding, we
constructed four sets of mutations that combined several single amino
acid substitutions. Mutants c1, c2, c3, and c4 each had four residues
substituted with alanine. They were residues 106, 114, 119, and 130 for
mutant c1; residues 107, 114, 121, and 130 for mutant c2; residues 106, 114, 121, and 130 for c3; and residues 107, 114, 119, and 130 for c4.
Sensorgrams for these multiply substituted proteins, with the exception
of c1, were also obtained by injecting 14 nM rhIL-18. Because c1 bound to rhIL-18
very poorly, 111 nM rhIL-18 was
injected. Except for c1, which cannot be compared with the others, the
association phases were similar to the unmutated hIL-18BP. There were,
however, differences in the dissociation phases. Residues at 107, 121, and 130, as a group, contributed very little to IL-18 binding because
mutant c2 only had a small increase in the dissociation rate compared
with that of the E114A mutant.
For kinetic analysis, sensorgrams that were obtained with various
concentrations of rhIL-18 and rmIL-18 were globally fitted with
BIAEVALUATION software to a 1-to-1 binding model, in which one molecule
of IL-18 binds with one molecule of hIL-18BP. Both the actual data and
fitted curve are shown for the mutated proteins with alanine
substitutions at amino acids 93 and 97 (Fig.
4). Similar experiments were performed at
least four times with each of the mutated proteins, and the kinetic and
affinity constants that were obtained are listed in Table
I. The relative changes of Gibbs free
energy change ( Crystal structures of protein-protein complexes usually reveal a
large interface, suggesting the involvement of many side chains that
are collectively called structural epitopes. However, the number of
side chains actually shown to be important for binding by alanine
mutagenesis is usually small. The latter amino acids are called the
functional epitopes. We mutated 15 residues in the hIL-18BP which
either had been predicted to be at the interface with IL-18 based on
the crystal structure of the related IL-1 and has an
important role in defense against microbial invaders. As its name
implies, the hIL-18BP binds to IL-18 with high affinity and prevents
the interaction of IL-18 with its receptor. We genetically modified the
C terminus of hIL-18BP by appending a 15-amino acid biotinylation
recognition site and a six-histidine tag and then performed
site-directed mutagenesis to determine the functional epitopes that
mediate efficient binding to IL-18. The mutated IL-18BPs were secreted from mammalian cells, captured by metal affinity chromatography, biotinylated in situ, eluted, and immobilized on
streptavidin-coated chips. Using surface plasmon resonance, we
identified seven amino acids of hIL-18BP which, when changed
individually to alanine, caused an 8-750-fold decrease in binding
affinity, largely because of increased off-rates. These seven amino
acids localized to the predicted
-strand c and
d of hIL-18BP immunoglobulin-like domain, and most had
hydrophobic side chains. Just two amino acids, tyrosine 97 and
phenylalanine 104, contributed ~50% of the binding free energy.
Information obtained from these studies could contribute to the design
of molecular antagonists of IL-18 for treatment of inflammatory diseases.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-inducing factor (1, 2). Acting synergistically with
costimulants such as IL-12, IL-18 induces interferon-
production
from T lymphocytes and macrophages, enhances the cytotoxicity of
natural killer cells, is required for an effective T helper-1 response,
and protects against bacterial and viral infections (3). In addition,
IL-18 has been implicated in endotoxin-induced liver damage and some chronic inflammatory diseases (4, 5). Based on its amino acid sequence
and predicted secondary structure, IL-18 was recognized as a member of
the IL-1 family (6). The similarity to IL-1
extends to the
processing of the IL-18 precursor by the IL-1
-converting enzyme (7,
8). In addition, the ligand binding and signaling subunits of the
heterodimeric IL-18 receptor are homologous to the corresponding
subunits of the IL-1 receptor, and all but the signaling subunit of the
IL-1 receptor map to a gene cluster in chromosome 2 (9-12).
Functionally, IL-18 signals through a pathway similar to that of IL-1,
which involves IL-1 receptor-activating kinase, TRAF6, and nuclear
factor-
B (13, 14).
(23). A model of hIL-18BP complexed to IL-18,
based on the IL-1R1·IL-1
structure, has been proposed (18).
To examine the structure/function relationships of hIL-18BP, we
performed site-directed mutagenesis. The binding affinity of each
mutated hIL-18BP for IL-18 was measured, and the residues important for interactions with IL-18 were determined.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
predicted that the
-strands c
and d, the loop between c and d, and
the f-g loop of hIL-18BP interact with IL-18
(18). We selected amino acid residues 93, 95, 97, 114, 130, 151, and
153 of hIL-18BP for mutagenesis because they were the ones suggested by
the model to interact with hIL-18 (Fig.
1). We also compared the amino acid sequence of hIL-18BP with those of two poxvirus-encoded IL-18BPs, MC54L
from molluscum contagiosum virus and evp16 from ectromelia virus. The
three IL-18BPs share sequence similarities in their Ig domains,
especially in the predicted
strands c, d, and
f (Fig. 1). Most of the conserved residues were also
selected for mutagenesis. Those not already chosen on the basis of the
structure model included residue 99 in the predicted c
strand, residues 104, 106, 107, 108, and 112 in the predicted
d strand, and residue 119 and 121 in predicted e
strand or d-e loop. Some other conserved residues
in the predicted f and g strands were not
selected for mutagenesis because they are distant from the predicted
interface between hIL-18BP and IL-18. Residue 121 of hIL18BP was
selected, although there was no corresponding residue in evp16. Among
these 15 residues chosen for mutagenesis (Fig. 1), 6 have charged side chains that could be involved in electrostatic interactions, and the
others have side chains that could participate in hydrophobic interactions.
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Fig. 1.
Multiple alignment of the Ig domains of
hIL-18BP, MC54L, evp16, and IL-1R1. The multiple alignment was
performed with the ClustalW program in the MacVector computer software
package and adjusted manually. Amino acids that are identical or
similar in three or more proteins are boxed. The
dark and light shading indicates amino
acids that are identical or similar, respectively. The lines
and the characters underneath the alignment indicate the
locations and the names of the -strands in IL-1R1 crystal structure.
The numbers above the alignment indicate the residue numbers
of hIL-18BP which are mutated in this study.
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Fig. 2.
Structure and expression of hIL-18BP fusion
protein. A, schematic representation of the C-terminal
fusion vector pYX45. The multiple cloning site (MCS),
flexible linker, biotinylation tag sequence, and six-histidine
(6Xhis) are boxed. The positions of the
restriction endonuclease sites and the stop codon are indicated above
the boxes, and the encoded amino acid sequences are shown
underneath. The lysine to which biotin can be added by biotin
holoenzyme synthetase is also indicated. B, schematic
representation of the constructs that were used for hIL-18BP
site-directed mutagenesis. The dashed and solid
lines represent hIL-18BP coding sequence. The lengths of the
dashed lines are not scaled to the length of the sequences.
The white box represents the sequence that was deleted in
the construct. The residues that were mutated are shown with their
residue number underneath. The unique KpnI site created in
the construct is indicated along with the unique NheI and
BamHI sites. The positions of PCR primers used for
mutagenesis are indicated along with the restriction enzymes. The
black solid boxes in the primers represent the amino acid
substitutions. C, detection of in vitro
biotinylated hIL-18BPs. Recombinant hIL-18BPs were bound to
Ni-nitrilotriacetic acid resin resin and biotinylated on the resin by
biotin holoenzyme synthetase. The proteins were eluted from the resin
with imidazole, and the biotinylated species were detected by
chemiluminescence with a streptavidin-horseradish peroxidase conjugate
after Western blotting. The positions and masses in kDa of protein
markers are shown on the left.
-strand c
and d caused increased rates of dissociation (Fig. 3,
A-D). Among these, substitution of the tyrosine at residue
97 or the phenylalanine at residue 104 caused the largest change.
Alanine substitutions at residues 93, 99, 106, 108, or 114 caused
smaller but significant increases in the off-rates (Fig. 3,
A-D). Outside of the predicted c and
d strands, no single mutation was found to affect the
binding significantly (Fig. 3E).
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Fig. 3.
Sensorgrams showing the binding of
mutated hIL-18BPs to hIL-18. The mutated hIL-18BPs were
biotinylated and captured individually on streptavidin chips. The
injection of rhIL-18 started at 150 s and stopped at 750 s.
The rhIL-18 concentration used was 14 nM in all cases
except c1, where it was 111 nM. The colored
lines are the responses obtained with the mutated hIL-18BPs and
normalized to a maximum of 100 RU. A and B,
single substitution at positions corresponding to strand c
of IL-1R1. C and D, single substitution at
positions corresponding to strand d of IL-1R1. E,
single substitution at all other positions. F, multiple
substitutions of charged amino acids.
G) caused by the mutations are also
shown in the table. Alanine substitution of the tyrosine at residue 97 or the phenylalanine at residue 104 increased the Kd
more than 100-fold above that of the unmutated hIL-18BP and together
contributed to ~50% of the binding energy. Alanine substitution of
residue 93 increased the Kd more than 50-fold and
contributed about 15% of the binding energy. In addition, substitutions of residues 99, 106, 108, and 114 all increased the
Kd by more than 5-fold. The substitution of lysine or glutamine for aspartic acid 114 caused a greater change than alanine
in the Kd.
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Fig. 4.
Kinetic analyses of the binding of IL-18BPs
with alanine substitutions at amino acid 93 or 97. The
biotinylated hIL-18BPs were captured individually on streptavidin
chips. Injection of rhIL-18 started at 150 s and stopped at
750 s. For the hIL18BP with alanine substitution at position 93, rhIL-18 concentrations were 0.22, 0.43, 0.87, 1.7, 3.5, 6.9, 14, and 28 nM. For the hIL-18BP mutated at position 97, rhIL-18
concentrations were 1.7, 3.5, 6.9, 14, 28, 56, and 111 nM.
The colored and black lines are the actual
responses in RU and globally fitted curves, respectively. The
residual responses, below each set of curves, represent
deviations of the actual responses from the fitted curves. The root
mean square deviation were 0.0833 for alanine 93 and 0.108 for
alanine 97.
Kinetics and affinity constants of hIL-18BP proteins
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
·IL-1R1 complex (18) or
were conserved in Ig domains of hIL-18BP and two viral IL-18BP homologs
(Fig. 5). When replaced by alanine, four
of the mutated hIL-18BPs exhibited more than a 5-fold decrease in
binding affinity with IL-18, and three exhibited more than a 50-fold
decrease. The effects on binding caused by the single alanine
substitutions are unlikely to have been caused by global changes in
IL-18BP structure for the following reasons. Alanine scanning
mutagenesis has been used successfully to probe many protein-protein
interfaces. Statistically, alanine is not a
-strand breaker and is
found in the middle of
-strands a, c,
f, and g of the IL-1R1 domain 3. In fact, most of
the alanine substitutions in IL-18BP caused small changes in affinity,
and those that produced large changes caused a small or no effect
on the on-rate. Furthermore, with the c2 and c4 mutations, the binding
free energy changes were almost exactly the sum of free energy changes
caused by the four individual substitutions. This result suggested that
the mutations did not cause major structural changes and that the residues 107, 114, 119, 121, and 130 interact independently with IL-18.
With the c1 and c3 mutations, the binding free energy changes were
somewhat greater than the sums of those caused by the individual substitutions. However, because c1 and c3 bound poorly to IL-18, the
surface plasmon resonance data did not have as good a fit to the 1-to-1
binding model as for the other mutations. We constructed structural
models for each mutated hIL-18BP based on the published wild type
hIL-18BP structural model (18). After energy minimization of the
mutated hIL-18BP structure models, the root mean square deviations of
the entire structure from the wild type structure were calculated (data
not shown). There was little difference in the deviations between those
mutations that caused more than a 5-fold change in affinity and those
that did not, consistent with the absence of large structural
perturbations caused by alanine mutagenesis.
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Fig. 5.
Locations of the mutated hIL-18BP residues in
a predicted hIL-18BP/hIL-18 structure model. The model was
reported previously (18) and was based on the x-ray
structure of IL-1 ·IL-1R1 complex. The backbone of hIL-18BP is
red, and the backbone of hIL-18 is blue. The side
chains of all of the hIL-18BP residues that are mutated are also shown.
Those residues that cause more than 5-fold change in binding affinities
when mutated to alanine are yellow, and those that cause
less than 5-fold change in binding affinities when mutated are
cyan.
The sequence alignment and the results of our binding data together
suggest that the functional epitopes cluster in -strands c and d of the Ig domain and point their side
chains outward. Residues 93-99 of hIL-18BP were confidently predicted
by the PhD program (25) to form a
-strand, and identical or
conservative amino acids are present in corresponding positions of the
MC54L and evp16 IL-18BPs. In addition, these sequences align very well with the sequences that form the
-strand c of IL-1R1
domain 3. Like the c-strand of nearly all Ig domains, a
tryptophan is found at position 98 of hIL-18BP and at the corresponding
positions of MC54L, evp16, and IL-1R1. In all of the Ig domain
structures examined, this conserved tryptophan points its side chain
inward, which is thought to be crucial for folding (26). As a result, residues 93, 95, 97, and 99 would point their side chains outward in a
-strand. This is consistent with our finding that residues 93, 97, and 99 all contributed significantly to binding, with residue 97 contributing around 25% of the binding free energy. A model, based on
the known structure of the complex of IL-1
and IL-1R1, predicted
that amino acids 93-99 of IL-18BP are in the
-strand and that
residues 93, 95, and 97 would be important for binding to IL-18 (Fig.
5). Our results minimized the role of residue 95.
Compared with the confidence in predicting the locations of residues
93, 97, and 99 in a three-dimensional structure, the locations
of residues 104, 106, 108, and 114 are less certain. This is because
different Ig domains vary considerably in the length and the sequences
of amino acids between the c- and e-strands (27).
In our multiple alignment of the Ig domains of IL-18BPs and IL-1R1
domain 3, there is a gap of seven amino acids between residue 103 and
104 of hIL-18BP. This gap was created because the sequences before and
after the gap are similar among the three IL-18BPs and because the
additional seven amino acids in evp16 and IL-1R1 share three identical
residues. This gap suggests that the -turn between the c-
and d-strands of IL-1R1 domain 3 is maintained in evp16 but
is shortened in hIL-18BP and MC54L. Residues 103-107 and 111-115 of
hIL-18BP were also predicted by the PhD program to form
-strands. We
suggest, therefore, that the d-strand of hIL-18BP includes
residues 103-115. This strand may be broken in the middle by proline
at 109 and glycine at 110. This is similar to the a-strand
of IL-1R1 domain 3, which has a proline in the middle of the strand
which breaks the strand into two smaller but parallel strands. Residues
104, 106, 108, and 114 of the hIL-18BP all contribute significantly to
the binding, suggesting that they face outward, whereas residue 107 does not. Our assignment of residues 104, 106, and 108 to the
d-strand is different from the previous prediction (18). In
that model, residues 102-110 form a
-turn that is similar in size
to the
-turn between the c- and d-strands of
IL-1R1 domain 3; consequently, residues 106 and 108 are located further
away from the interface with IL-18 and would not seem capable of making
contact with IL-18 (Fig. 5).
Phenylalanine 93, tyrosine 97, and phenylalanine 104 together
contributed ~65% of the free energy for binding of hIL-18BP to
IL-18. This is similar to the situation for human growth hormone binding to its receptor in which two tryptophans contribute
three-quarters of the binding free energy (28). Such "hot spots" in
protein-protein interactions frequently involve tryptophan, tyrosine,
and arginine (29). Phenylalanine is also involved, although with lower
frequency. Tryptophan and tyrosine can participate in hydrogen bonding,
-interactions, and van der Waals interactions, accounting for their
important role in protein-protein interactions. Phenylalanine can
participate in
- and van der Waals interactions but not hydrogen
bonds. The tendency of hot spot residues to be at the center of a
protein-protein interface reinforces the predicted locations of
residues 93 and 97 in the c-strand and residue 104 in the
d-strand. Our finding that a small number of residues of
hIL-18BP contributed the majority of the binding free energy suggests
that a small molecule mimicking the relative small functional epitopes
may be possible. One starting point for such an antagonist is a peptide
comprising the c- and d-strands of hIL-18BP which
have been constrained so as to bring them together side by side.
Alternatively, a phage display library with a bias toward the known
important residues may be used to find a peptide that binds tightly
with IL-18.
We developed a facile procedure for coupling hIL-18BP to sensor chips
which should be applicable to other recombinant proteins. The coding
sequence of hIL-18BP was modified to contain a C-terminal tail that
included a flexible linker, a biotin holoenzyme synthetase biotinylation recognition sequence, and a six-histidine tag. After transfection of mammalian cells with an expression plasmid, the secreted hIL-18BP was captured on metal affinity resin via the polyhistidine tag. Biotinylation was achieved by incubating the resin
with biotin holoenzyme synthetase and biotin, after which the hIL-18BP
was eluted with imidazole and directly bound to a streptavidin-coated
sensor chip. The coupling of the recombinant protein to the chip
surface via a single biotin attached to a flexible tail should allow a
uniform protein conformation and minimize steric hindrance to ligand
binding. It is possible that this improvement was responsible for the
Kd of 0.06 nM obtained here compared
with the higher value of 0.4 nM reported previously using
protocols in which nonspecific amine coupling was used to attach
hIL-18BP (17) or rhIL-18 (18) to the chip.
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ACKNOWLEDGEMENTS |
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We thank M. Rubinstein for providing the hIL-18BP and IL-18 coordinates of the published structural model (18), K. Ishii for 293T cells, and N. Cooper for maintenance of cell lines.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: NIAID, National
Institutes of Health, 4 Center Dr., MSC 0445, Bethesda, MD 20892-0445. Tel.: 301-496-9869; Fax: 301-480-1147; E-mail: bmoss@nih.gov.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M009581200
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ABBREVIATIONS |
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The abbreviations used are: IL, interleukin; hIL-18BP, human IL-18 binding protein; IL-1R1, type 1 IL-1 receptor; PCR, polymerase chain reaction; rmIL-18, recombinant murine IL-18; RU, response units; rhIL-18, recombinant human IL-18.
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