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IMMUNOBIOLOGY
Crystal structure and collagen-binding site of immune inhibitory receptor LAIR-1:
unexpected implications for collagen binding by platelet receptor GPVI
T. Harma C. Brondijk,1 Talitha de Ruiter,2 Joost Ballering,1,3 Hans Wienk,3 Robert Jan Lebbink,2 Hugo van Ingen,3
Rolf Boelens,3 Richard W. Farndale,4 Linde Meyaard,2 and Eric G. Huizinga1
1Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands; 2Department of Immunology,
University Medical Center Utrecht, Utrecht, The Netherlands; 3NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The
Netherlands; and 4Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
Leukocyte-associated immunoglobulinlike receptor-1 (LAIR-1), one of the most
widely spread immune receptors, attenuates immune cell activation when bound
to specific sites in collagen. The collagenbinding domain of LAIR-1 is homologous
to that of glycoprotein VI (GPVI), a collagen receptor crucial for platelet activation. Because LAIR-1 and GPVI also
display overlapping collagen-binding
specificities, a common structural basis
for collagen recognition would appear
likely. Therefore, it is crucial to gain insight into the molecular interaction of
both receptors with their ligand to prevent unwanted cross-reactions during
therapeutic intervention. We determined
the crystal structure of LAIR-1 and
mapped its collagen-binding site by
nuclear magnetic resonance (NMR) titrations and mutagenesis. Our data identify
R59, E61, and W109 as key residues for
collagen interaction. These residues are
strictly conserved in LAIR-1 and GPVI
alike; however, they are located outside
the previously proposed GPVI collagenbinding site. Our data provide evidence
for an unanticipated mechanism of collagen recognition common to LAIR-1 and
GPVI. This fundamental insight will contribute to the exploration of specific means
of intervention in collagen-induced signaling in immunity and hemostasis. (Blood.
2010;115:1364-1373)
Introduction
The leukocyte receptor complex (LRC) on human chromosome 19
encodes 2 receptors for collagens that are structurally related:
leukocyte-associated immunoglobulin (Ig)–like receptor-1 (LAIR-1)
and glycoprotein VI (GPVI). Their genomic location and structural
homology suggest a common origin for these 2 collagen-binding
receptors. However, their function is in 2 completely different
biologic systems and on mutually exclusive cell subsets.
LAIR-1 belongs to the family of immune inhibitory receptors,
which are involved in controlling the balance of the immune
system to prevent improper activation or overactivation, which
may result in tissue damage or autoimmune diseases. Upon
interaction with their ligands, these receptors attenuate the signals
provided by activating receptors, thereby increasing the threshold
for activation. LAIR-1 is one of the most widely distributed
inhibitory receptors, expressed on most immune cells, including
natural killer cells, T cells, B cells, monocytes, and CD34⫹ hematopoietic progenitor cells.1-5
LAIR-1 is a type I glycoprotein of 287 amino acids containing a
single extracellular Ig-like domain followed by a stalk region
connected to the single transmembrane domain and 2 cytoplasmic
immunoreceptor tyrosine-based inhibitory motifs that relay the
inhibitory signal.2 LAIR-1 binds both transmembrane and extracellular matrix collagens.6,7 Under physiologic conditions, immune
cells present in the bloodstream do not encounter matrix collagens.
However, their extravasion results in exposure to the collagen-rich
subendothelium, where LAIR-1 transduces an inhibitory signal to
the cell, resulting in abrogation or inhibition of immune cell
function.6
As opposed to LAIR-1, GPVI is an activating receptor, with 2
instead of 1 extracellular Ig-like domain. GPVI is expressed by
megakaryocytes and platelets and plays a central role in hemostasis
by activating platelets upon binding to collagen, along with von
Willebrand factor and the collagen-binding integrin ␣2␤1.8,9
Collagens play crucial roles in the development, morphogenesis, and growth of many tissues. Their role in hemostasis is well
established and the recent identification of LAIR-1 as an inhibitory
collagen receptor reveals a novel role for collagen as immune
regulatory protein.1,6 All collagens are composed of 3 polypeptide
chains that are characterized by a repeating Gly-X-X⬘ sequence,
where X is often proline and X⬘ frequently 4-R-hydroxyproline
(Hyp, O). The GPO triplets are an almost exclusive feature of
collagens and allow the formation of the characteristic triplehelical collagen structure. Although GPVI and LAIR-1 are functionally different, they are similar in their collagen-binding properties.
Both receptors can bind GPO repeats.10 When expressed on the
same cell, LAIR-1 is capable of inhibiting collagen-induced GPVI
signaling.11 Recently, collagen-binding properties of GPVI and
LAIR-1 have been characterized using collagen II– and collagen
III–derived synthetic Toolkit peptides.7,12 LAIR-1 appears to have
several binding sites on collagens II and III and its binding
spectrum on the latter is overlapping, although not identical, with
that of GPVI.13
Submitted October 2, 2009; accepted November 13, 2009. Prepublished online
as Blood First Edition paper, December 10, 2009; DOI 10.1182/blood-2009-10246322.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2010 by The American Society of Hematology
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BLOOD, 18 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 7
LAIR-1 and GPVI are structurally related to several other
inhibitory and activating LRC receptors. These include leukocyte
inhibitory receptors, such as LIR-1, LIR-2 and LILRB5, the
killer-cell Ig-like receptors (KIRs), and the IgA receptor Fc␣RI.
The extracellular domain of LRC receptor proteins consists of
1 (LAIR-1), 2 (GPVI, LIR-5, Fc␣RI, p58 KIRs), 3 (p70 KIRs), or
4 (LIR-1, LIR-2) Ig-like domains, termed D1 to D4. The ligandbinding sites of these receptors are limited to the D1 domain and
the D1D2 hinge region. The crystal structures of the D1 to D2
region of several LRC receptors in complex with their respective
ligands have been solved.14-22 Despite large structural overlaps,
LRC receptors display a remarkable versatility in the organization
of their ligand recognition site (supplemental Figure 1, available on
the Blood website; see the Supplemental Materials link at the top of
the online article).
Manipulation of either thrombosis through GPVI or the immune
system through LAIR-1 can be envisaged using reagents that
interfere with collagen binding of either one of these receptors.
However, to prevent unwanted side effects resulting from interference with the other receptor, it is important to precisely characterize their collagen-binding sites. The large variation in ligand
interaction sites of LRC receptor proteins and the limited data on
the GPVI/collagen interaction make it difficult to predict the
LAIR-1 collagen-binding site and emphasize the need for more
structural information for this family of proteins.
In this paper, we present the 1.8-Å crystal structure of the
human LAIR-1 collagen-binding domain. We mapped the collagenbinding site to the structure using nuclear magnetic resonance
(NMR) titrations and site-directed mutagenesis. The LAIR-1
collagen-binding site is shown to be surprisingly different from the
proposed GPVI collagen-binding site.
Methods
Expression and purification
hLAIR-1a cloned into the pMX retroviral vectors was described previously.6 Site-specific mutations were introduced using the QuikChange
method (Stratagene) and confirmed by sequencing. Wild-type and mutant
hLAIR-1 were stably expressed in human erythroleukemia K562 cells by
retroviral transduction as described previously.6 The isolated collagenbinding domain of hLAIR-1 (hLAIR1-CBD), amino acids 22 to 122, was
expressed in Escherichia coli and purified by affinity chromatography and
size exclusion chromatography as described in the supplemental data.
Crystallization and crystal structure determination
Crystals of hLAIR1-CBD were grown using vapor diffusion at 18°C in 25%
to 30% polyethylene glycol-1500, 100mM succinic acid, 100mM sodium
dihydrogen phosphate, 100mM glycine, 10mM CoCl2, pH 4.1 to 4.4.
Crystals appeared within 0.5 to 2 days. Protein concentration before
setting up the drops was 20 to 30 mg/mL. For cryoprotection, crystals
were transferred to the crystallization solution supplemented with 20%
(vol/vol) glycerol.
Diffraction data were collected at 100 K at beamline ID23-1 of the
European Synchrotron Radiation Facility and processed with Mosflm.23
The structure was solved by molecular replacement with the programs
PHASER and MolRep, which are part of the CCP4 suite,23 using a model
generated by the Swiss-model server24 based on an alignment of LAIR-1
with GPVI, LILRA5, LIR-1, and LIR-2 (Protein Data Bank entries 2GI7,
2D3V, 1UGN, and 2GW5, respectively).25 Initial rounds of refinement were
done using Refmac5,23 followed by twin least squares-refinement in
Phenix.26
The final model consists of 3 molecules hLAIR1-CBD and contains 1
glycine and 1 glycerol molecule. Stereochemistry was checked using
STRUCTURE AND COLLAGEN-BINDING SITE OF LAIR-1
1365
Molprobity.27 Data collection and refinement statistics are listed in supplemental Table 1. Graphics were generated using PYMOL (DeLano Scientific). The final coordinates were deposited at the Protein Data Bank under
accession code 3KGR.25
NMR experiments
15N-labeled hLAIR1-CBD was produced as described in the supplemental
data. The collagen III–derived synthetic peptide III-30 was synthesized as
described previously.28 The amino acid sequence of a single chain of the
peptide is GPC(GPP)5GAOGLRGGAGPOGPEGGKGAAGPOGPO(GPP)5GPC-NH2 with bold residues denoting the collagen III–derived
sequence. The free cysteines were blocked by S-carboxymethylation using
0.50mM iodoacetamide at 37°C. The peptide was transferred to 10mM
potassium phosphate, pH 7.1, using a High-Trap desalting column (GE
Healthcare) and concentrated to 3.1mM.
NMR experiments were performed on a Bruker Avance III spectrometer operating at 600-MHz proton resonance frequency. For spectral
assignment 15N-NOESY–heteronuclear single quantum coherence
(HSQC) and 15N-TOCSY-HSQC spectra were recorded at 25°C with
0.8mM 15N-hLAIR in 10mM potassium phosphate buffer, pH 7.2,
containing 10% D2O. For the titration,{1H;15N}-HSQC spectra at 30°C
followed the stepwise addition of small amounts of the collagen III-30
peptide to 0.1mM 15N-LAIR-1. hLAIR-1/triple-helical collagen III-30
ratios varied between 1:0.1 and 1:3.7.
All spectra were processed using NMRPipe29 and analyzed with Sparky
(T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San
Francisco). Data are deposited at the Biological Magnetic Resonance Bank
under accession code 16591.30
Collagen interaction
Interaction of hLAIR1-CBD with collagen was confirmed in a solid-state
binding assay. Microtiter wells were coated with collagen I or collagen III
(50 ␮g/mL) in phosphate-buffered saline (PBS; pH 7.4) for 16 hours at
4°C. After washing, wells were blocked with 3% bovine serum albumin
(BSA) and subsequently incubated with serial dilutions of hLAIR1-CBD in
PBS containing 0.01% Tween-20 and 2 mg/mL BSA for 2 hours at room
temperature (RT). After washing, anti–human LAIR-1 antibody (clone
DX26) was added to the plates for 1 hour at RT. After washing 3 times with
PBS/0.01% Tween-20, wells were incubated with horseradish peroxidase–
conjugated polyclonal goat anti–mouse antibodies for 1 hour at RT. After 3
washes with PBS/0.01% Tween-20, bound antibodies detecting LAIR-1
were visualized via incubation with 2,2⬘-azino-bis(3-ethylbenzthiazoline-6sulphonic acid.
LAIR-1/collagen interactions in solution were assayed by incubating
K562 cells transfected with wild-type or mutant hLAIR-1a with fluorescein
isothiocyanate (FITC)–conjugated collagen I or collagen III (conjugated as
described in Van de Walle et al31) or Oregon green 488–conjugated collagen
IV (Molecular Probes) plus phycoerythrin-conjugated anti–LAIR-1 monoclonal antibody (BD Biosciences). Cells were incubated for 30 minutes,
washed, and analyzed by flow cytometry (FACSCalibur; BD).
Binding of K562 transfectants to plate-bound collagen was analyzed by
adhesion of calcein-labeled cells; 96-well MAXIsorp flat-bottom plates
were coated overnight at 4°C with collagen I, III, or IV (10 ␮g/mL;
Sigma-Aldrich) or BSA (5 ␮g/mL) in 100 ␮L of PBS supplemented with
2mM acetic acid. Wild-type K562 or K562 cells (5 ⫻ 106/mL), stably
transfected with hLAIR-1a, were assayed for their capacity to adhere to the
collagens in the 96-well plates as described before.6
Docking calculations
Three-dimensional models of the LAIR-1/peptide III-30 complex were
calculated using the HADDOCK web server (http://haddock.chem.uu.nl/
Haddock/haddock.php, Utrecht University). Details of model construction
and the docking procedure are described in the supplemental Data.
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BLOOD, 18 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 7
BRONDIJK et al
A
Figure 1. LAIR-1 overall structure and comparison with other
LRC-family members. (A) Ribbon drawing of the LAIR-1 ectodomain structure in rainbow colors from N-terminus (blue) to Cterminus (red). The disulfide bond between ␤-strands B and F,
characteristic of Ig-like domains, is indicated in stick representation.
(B) Superposition of the LAIR-1 ectodomain (gray) with the D1
domains of LRC-family members KIR2DL2, GPVI, LIR1, and the
Fc-alpha receptor, respectively (red).
B
C
F
C’
B E
KIR2DL2
G VI
GpVI
A
G
A’
310
LIR1
Results
The isolated collagen-binding domain of LAIR-1 retains its
functionality
The collagen-binding domain of human LAIR-1 (hLAIR1-CBD)
was expressed in E coli. The purified CBD bound to immobilized
human collagens I and III with apparent dissociation constants of
147nM (⫾ 16nM) and 750nM (⫾ 71nM), respectively (supplemental Figure 2). The hLAIR1-CBD also displayed significant binding
to GPO10, albeit, like the native protein,6 with much reduced
affinity compared with collagen (data not shown). These apparent
dissociation constants are lower than the previously determined
dissociation constant of 26␮M for collagen I binding of hLAIR1CBD fused to rat CD4 domains 3 and 4,32 whereas a hLAIR-1-IgG
fusion displays even lower dissociation constants of 14.2nM
(⫾ 2.7nM) and 16.2nM (⫾ 3.8nM) for collagens I and III, respectively.6 Stronger binding of the hLAIR-1-IgG fusion is explained
by its dimeric nature, arising from disulfide bridge formation
between the IgG parts. The relatively high affinity of our monomeric E coli–expressed hLAIR1-CBD for collagens I and III
strongly suggests that it is properly folded and shows that the single
N-glycosylation site at residue N692 that remains unglycosylated
upon expression in E coli is not essential for collagen binding.
Crystal structure
The hLAIR-1 collagen-binding domain was crystallized and its
structure was solved to 1.8-Å resolution using molecular replacement (supplemental Table 1). The asymmetric unit of the crystal
contains 3 hLAIR1-CBDs that were built and refined independently. The 3 CBDs are essentially identical with main chain root
mean square difference (rmsd) values ranging from 0.28 to 0.39 Å.
The LAIR1-CBD forms a single I-type Ig-fold consisting of a
sandwich of 2 ␤-sheets composed of ␤-strands ABE and A⬘GFCC⬘
(Figure 1A). A disulfide bond between ␤-strands B and F connects
the 2 ␤-sheets. The A⬘ strand is created by a cis-proline (P35) that
introduces a sharp bend after the first ␤-strand. The loop between
␤-strands E and F contains a short 310-helix. The N-linked
glycosylation site at N69 is located in ␤-strand C⬘ and is fully
exposed to the solvent.
FcαR
The overall structure of the hLAIR1-CBD is similar to the D1
domains of other LRC receptors with C␣ rmsd values ranging from
1.1 to 2.1 Å (Figure 1B and supplemental Table 2). The largest
differences between these proteins are found in the CC⬘-loop, the
C⬘-strand, and the C⬘E-loop region. In LIR-1 and LIR-2 strand, C⬘
is replaced by a 310-helix. In GPVI, there is a 12–amino acid
deletion in this region, including part of ␤-strands C⬘ and E and
most of loop C⬘E. The closest structural matches to LAIR-1 are the
KIR2DL2, LILRA5, and GPVI D1 domains with C␣ rmsd values
of 1.34 Å over 93 residues, 1.36 Å over 91 residues, and 1.09 Å
over 85 residues, respectively.
LAIR-1 signaling is most likely induced by multimerization as
cross-linking using monoclonal antibodies results in receptor signaling
and cell down-regulation.2,3,6,33 Inspection of the crystal packing reveals
that 2 of the 3 CBD molecules in the asymmetric unit form an
antiparallel dimer, whereas the third molecule does not share an
extended interface with any other molecule in the crystal (supplemental
Figure 3). We analyzed the potential biologic significance of the dimer
using the PISA web server (European Bioinformatics Institute).34 On the
basis of the relatively small size of the dimer interface (439 Å2) and the
nature of the interaction surface, PISA suggests that the dimer observed
in the crystal is not likely to be of physiologic relevance.
LAIR-1 and GPVI bind to an overlapping set of synthetic
collagen peptides.7,12 It is therefore tempting to speculate that their
collagen-binding sites are similar. Based on the GPVI crystal
structure, mutagenesis data and rigid body docking calculations, it
has been proposed that the collagen-binding site in GPVI is a
shallow hydrophobic groove formed by ␤-strands C⬘ and E.14
Although these strands are more extended in LAIR-1, also here a
groove is found at this position. Interestingly, R65, a residue
recently shown to be important for LAIR-1 collagen binding,35 is
located at one end of this groove. Furthermore, rigid body docking
calculations using the PatchDock web server (Tel Aviv University)
placed a collagen peptide in this groove. To obtain experimental
data on the location of the collagen-binding site of LAIR-1, we
performed NMR spectroscopy and mutagenesis studies.
Mapping the collagen-binding site of hLAIR1-CBD by NMR
Previously, screening of collagen II and collagen III peptide
libraries has identified several triple-helical peptides as functional
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BLOOD, 18 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 7
ligands for LAIR-1.7 We used the most potent of these, peptide
III-30 (see “NMR experiments”), to map the collagen-binding
region in 15N-labeled hLAIR1-CBD by NMR titration experiments.
{1H;15N}-HSQC spectra were recorded in the absence and in the
presence of increasing amounts of peptide III-30. In absence of
ligand, the CBD shows a well-dispersed spectrum of homogenous
line-width, indicative of a single well-defined fold (Figure 2A).
Most of the backbone and side chain amides were assigned
unambiguously using 15N-TOCSY-HSQC and 15N-NOESY-HSQC
spectra. The most N-terminal residues, G20-Q22, residues S64-R65S66 in the C-C⬘ loop, and G94 remain unassigned. Two signals in
the HSQC spectrum most likely belong to residues from the
S64-S66 loop. However, their assignment remains ambiguous due
to low signal intensity.
Titration of peptide III-30 resulted for all cross-peaks in a
gradual increase in line-width due to the substantial increase in
mass upon binding of the 11.5-kDa hLAIR1-CBD to the 16.0-kDa
triple-helical peptide. Besides that, we observed clear shifts of
several cross-peaks (Figure 2B). Signals of residues that have
different chemical shifts in the free and bound state of hLAIR1CBD experience additional line broadening due to continuous
interconversion between the 2 states on the NMR time scale. For
relatively small chemical shift differences (ie, limited influence of
peptide binding), the residues are in fast-to-intermediate exchange
and the peaks can be followed throughout the titration. For residues
that show relatively large chemical shift differences (intermediateto-slow exchange), line broadening is severe, and the signal is lost
in the course of the titration. Overall, this observed spectral
behavior is consistent with a collagen-binding affinity in the
micromolar range.
The signals that shifted more than one line-width and displayed
intermediate-to-slow exchange were considered to constitute the
peptide III-30 binding interface on hLAIR1-CBD. Residues showing this behavior are R59-E63, T67-N69, A84-F86, R100-I102,
W109-S113, Y115-L116, and L118. In addition, the side chain
resonances of W109 and Q112 are affected by the presence of
collagen III-30, and, finally, although this region could not be
assigned, peptide binding affects probably one of the unassigned
residues in the sequence S64-R65-S66. The residues that are
perturbed by peptide addition cluster on ␤-strands C⬘, C, and F, and
form a continuous area on the surface (Figure 2C). The location of
the LAIR-1 collagen-binding surface as identified by NMR resembles the major histocompatibility complex I–HLA interaction
sites of LIR-1 and LIR-2,15,19 rather than the proposed collagenbinding site in GPVI (compare Figure 2C and supplemental Figure 1).
Collagen-binding site mutants
Assignment of the hLAIR-1 collagen-binding surface was verified
by site-directed mutagenesis. We designed 13 mutations, including
5 in the putative collagen-binding site as indicated by the NMR
titration experiments, 2 in the unassigned loop S64-S66, and 6
outside the region with the largest chemical shift changes, of which
4 correspond to the putative collagen-binding site of GPVI.
Mutant hLAIR-1 proteins were stably expressed in K562 cells
and tested in a flow cytometric assay for binding to FITCconjugated collagens I and III or Oregon green 488–conjugated
collagen IV (Figure 3A-B). Surface expression levels of the mutant
proteins, as determined by binding of LAIR-1 antibodies (clones
DX26 and 8A8) in the flow cytometric experiments were comparable with the wild-type protein (Figure 3A), which is an indication
of proper folding. Mutants R59A, E61A, and R65A showed
severely reduced binding to all collagens tested. In addition, mutant
STRUCTURE AND COLLAGEN-BINDING SITE OF LAIR-1
1367
E111A showed decreased collagen binding, albeit to a lesser extent.
The R62A mutant displayed somewhat reduced binding to collagen
III, even further reduced binding to collagen IV, and unaffected
binding to collagen I. None of the mutations outside the area
designated by the NMR experiments significantly affected collagen
binding. We also measured the adhesion of transfected cells to
immobilized collagens for a subset of LAIR-1 mutants (Figure 3C).
The subset included all 5 mutants that showed decreased collagen
binding in the flow cytometric assay and 4 randomly selected
mutants that displayed unaffected collagen binding. Adhesion to
immobilized collagens I, III, and IV was significantly reduced in
the R59A, E61A, R65A, and E111A mutants, although the
magnitude of the effect depended on the type of collagen tested. In
addition, adhesion was somewhat reduced for mutants R62A and
N69A, which showed near wild-type collagen binding in the flow
cytometric assay.
Binding of hLAIR1 mutants to FITC-conjugated peptide III-30
in the flow cytometric assay was severely decreased for the R59A
mutant, slightly reduced for the E61A and E111A mutant proteins,
and unaffected for all other mutant LAIR-1 proteins except the
N69A mutant. Comparable results were obtained with the adhesion
to coated peptide III-30 (supplemental Figure 4). Surprisingly, the
R65A and E61A mutations do not or only slightly affect peptide
III-30 binding, although both mutations severely reduce binding to
intact collagens. Apparently, recognition of a specific binding site
in the context of the triple-helical peptide III-30 differs somewhat
from binding to intact fibrous collagens (see “Discussion”).
The mutational analysis confirms the location of the LAIR-1
collagen-binding site as determined by the NMR titration (compare
Figure 2B and Figure 4A) and establishes that residues R59,
E61, R65, and E111 contribute significantly to the affinity of
collagen binding.
Docking of LAIR-1 and collagen model peptides
We combined our crystallographic, NMR, and mutagenesis data to
generate models of the LAIR-1/collagen complex by data-driven
docking using the program HADDOCK.36-38 Docking of the
LAIR1-CBD crystal structure to a theoretic model of peptide III-30
was driven by intermolecular distance restraints between the
collagen peptide and CBD residues that were identified by NMR
and/or mutagenesis to be close to or in the binding site. As no
experimental data on interacting residues from collagen are available, all surface-accessible residues of the collagen models were
allowed to participate in docking.
The 2 best scoring solutions show the collagen peptide bound at
an angle of roughly 60° to the F, C, and C⬘ ␤-strands of LAIR-1
(Figure 4B). These solutions have similar Haddock scores, well
above the scores of other solutions (data for the best 10 solutions
are presented in supplemental Table 3). Whereas the interaction site
on LAIR-1 is very similar in both solutions, binding occurs to
different regions of the collagen peptide: the C-terminal sequence
KGAAGPOGPO or the more N-terminal sequence RGGAGPOGPE. Both putative binding sites comprise residues with short
side chains including 2 and 1 GPO motif. Although this could
reflect an intrinsic binding preference of LAIR1, we cannot exclude
that it is an artifact of the docking procedure. Binding at 2 sites
would be consistent with the suggested presence of 2 binding sites
in peptide III-30,8 with the most important binding site for receptor
signaling located toward the C-terminal 2 GPO triplets. However,
due to the limited experimental data on collagen residues
involved in LAIR binding, the docking results should be
interpreted with caution.
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BLOOD, 18 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 7
BRONDIJK et al
A
B
C
E111
W109
I102 R59
Q112
E61 Y68
R100
N69
T67
R65
Figure 2.
180˚
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BLOOD, 18 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 7
Figure 3. Binding of K562 cells transfected with
hLAIR-1a mutants to various collagens. (A) Flow
cytometric analysis of LAIR-1 expression (y-axis) and
FITC-conjugated collagen I binding (x-axis) on parent
K562 cells (upper left panel) or K562 cells expressing wt
or mutant LAIR-1a as indicated. Representative dot plots
of at least 3 independent experiments are shown. Percentage of LAIR-1⫹ collagen-binding cells is indicated.
(B) Summary flow cytometric analyses showing binding
of soluble collagens I, III, and IV to parent K562 cells (⫺)
and K562 cells expressing wt LAIR-1a or mutants as
indicated. (C) Adhesion of parent K562 cells (⫺) and
K562 cells expressing wt LAIR-1a or mutants as indicated
to plate-bound collagens I, III, and IV. White bars indicate
collagen I; black bars, collagen III; and striped bars,
collagen IV (nt indicates not tested). Data represent
mean ⫾ SD of at least 3 independent experiments.
STRUCTURE AND COLLAGEN-BINDING SITE OF LAIR-1
1369
A
B
C
Variations between solutions of comparable scores are considerable (Figure 4B), nevertheless, some general conclusions seem
justified. The peptide binds across the ␤-sheet, burying a surface
area of approximately 1300 Å2. The binding site comprises
3 collagen triplets, with LAIR-1 contacting 2 of the 3 collagen
peptide chains. LAIR-1 residues E61, S66, Y68, I102, W109, and
Y115 provide Van der Waals interactions, whereas hydrogen bonds
to the ligand frequently involve LAIR-1 residues R59, E63, R100,
E111, and Q112 (Figure 4C). In our docking solutions, we see no
significant interactions involving the R65 side chain; however, it is
in close enough proximity to the peptide to envisage an interaction
upon a minor and local conformational change. The weak intensities of peaks attributed to residues S64-R65-S66 in the NMR
experiments indicate the presence of slow dynamics in this loop
region that would enable R65 to adopt multiple conformations,
which may not be sampled during docking calculations. Our
docking model gives insight into the mechanism by which LAIR-1
and collagen interact and provides a basis for further mutagenesis
studies on both the collagen peptide and LAIR-1.
Discussion
Collagen interactions are essential in many biologic processes
including immune suppression, cell migration, and hemostasis.
Here we set out to explore the interaction of LAIR-1 with collagen
using a combination of X-ray crystallography, NMR titrations, and
mutagenesis. The crystal structure of the collagen-binding domain
of hLAIR-1 was determined to 1.8-Å resolution and most closely
resembles the KIR2DL2, LILRA5, and GPVI D1 domains. The
Figure 2. NMR analysis of the titration of unlabeled collagen III-30 peptide to 15N-labeled hLAIR1-CBD. (A) Assigned {1H;15N}-HSQC spectrum of the isolated LAIR-1
ectodomain in the absence of ligand. Gray lines connect the resonances of asparagine and glutamine side chain amides. The boxed region contains arginine side chain
resonances. Side chain assignments are indicated by sc. Unassigned signals are indicated by #. (B) Selected regions of the overlay of {1H;15N}-HSQC spectra recorded at
different peptide III-30 concentrations. Shown are representative examples of residues with large chemical shift changes that are in intermediate-to-slow exchange (R59, R62,
Y68, I102, S110, #), residues that are in fast-to-intermediate exchange (V74), and residues that show no shift (E24, V120) or shifts smaller than a line-width (E81, A96). The
arrows indicate the direction of peak displacement. Color coding reflects the relative peptide concentration as indicated. (C) Mapping of spectral changes on a ribbon
representation of hLAIR1-CBD (left), a surface representation in the same orientation (middle), and a surface representation rotated by 180° around a vertical axis (right). White
indicates no data due to lacking assignments or spectral overlap; blue, no spectral change or change smaller than one line-width; orange, residues in fast-to-intermediate
exchange; and red, residues in intermediate-to-slow exchange. Residues of interest are labeled.
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BLOOD, 18 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 7
BRONDIJK et al
B
A
C
W109
E111
E111
R59
I102
E61
Q112
N69
R62
R65
R59
R100
Y104
E63
E61
Y68
S66
Figure 4. The LAIR-1/collagen complex. (A) Surface representation of LAIR-1 showing the effect of mutations on collagen binding: red indicates strong decrease; pink,
moderate decrease; orange, moderate decrease in the plate adhesion assay only; and blue, no effect. (B) Surface representation of LAIR-1 with ribbon representations of
collagen peptide III-30 models from the 2 highest scoring docking solutions (supplemental Table 3), shown in red and yellow. LAIR-1 residues that contact the docked peptides
are shown in green. (C) Close-up of the putative collagen-binding site, showing in sticks LAIR-1 residues that are in contact with the docked collagen peptide. Residues that
affect collagen binding when mutated are shown in blue. The collagen peptide is shown in semitransparent surface representation.
NMR data define a continuous surface patch on the hLAIR1-CBD
that is involved in the interaction with collagen. Probing of this
interaction surface by site-directed mutagenesis shows residues
R59 and E61 to be essential for collagen binding. The importance
of R59 and E61 is further emphasized by their strict conservation in
LAIR proteins from 9 different species (supplemental Figure 5).
Residues R59 and E61, together with W109, form a patch of
conserved surface-exposed residues in the core of the collageninteraction surface as identified by NMR (Figures 2B and 5C).
Although W109 was not probed by mutagenesis, the chemical
environment of its side chain changes upon collagen binding, as is
evident from the spectral shift of the side chain nitrogen-resonance
in the NMR titration. In combination with its strict conservation,
this is suggestive of an important role for W109 in ligand binding.
Mutagenesis data show a limited contribution to collagen binding
of residue E111, located at the periphery of the interaction surface.
Accordingly, E111 is not conserved. Surprisingly, R65 is also not
conserved, despite its essential role in collagen binding described
previously35 and confirmed in this study. Its important role, but lack
of conservation, indicates the existence of species-specific differences in collagen interactions. Also of note: mutation of R65 has no
effect on binding to peptide III-30 (further discussed below). We
postulate that residues R59, E61, and also W109 make up the core
of the collagen-binding site, whereas more peripheral residues such
as E111 contribute less to binding. The role of R65 in the
LAIR-1/collagen interaction remains ambiguous.
Comparison of ligand-binding sites of LRC-family members
shows that they are diverse and located at different positions on
the D1 and D2 domains, reflecting the diversity of ligands bound
by the various family members (supplemental Figure 1). Surprisingly, the location of the LAIR-1 collagen-binding site, across
the FCC⬘ ␤-sheet, most closely resembles the major histocompatibility complex–HLA interaction sites of LIR-1 and LIR-2 and
not the proposed collagen-binding site of GPVI, which is
located in a hydrophobic groove on the surface in between
␤-strands C⬘ and E.14
In view of their homology and overlapping ligand preferences,7,12 it is remarkable that the proposed LAIR-1 and GPVI
collagen-binding surfaces differ so completely. Mapping of conserved surface residues reveals, however, significant conservation
of the LAIR-1 collagen-binding site in GPVI (Figure 5 and
supplemental Figures 5-6). Conserved residues include R59, E61,
and W109, which are central to collagen binding in LAIR-1. The
converse is not true: residues implicated in the collagen-binding
site of human GPVI are not conserved in LAIR-1. Surprisingly, the
putative GPVI collagen-binding site is not conserved in GPVI
orthologues either (Figure 5B,E and supplemental Figure 6). This is
unexpected because protein-protein interaction sites in general
belong to the most conserved surface residues among orthologous
proteins. Identification of the GPVI collagen-binding site has relied
on rigid body docking of a collagen-like peptide on the crystal
structure of human GPVI and is supported by mutagenesis data on
GPVI residues, not conserved in LAIR-1: K59, R60, and R166.14,39,40
The collagen-binding mode that we propose for LAIR-1 would not
place these GPVI residues in the proximity of collagen and thus
cannot provide an alternative explanation for the GPVI mutagenesis data. It should be noted that some evidence suggests the
existence of 2 collagen-binding sites on GPVI or at least a more
extended collagen-binding site, including GPVI residues G30,
V34, and L38.41 It is possible that LAIR-1 and GPVI have evolved
different strategies for binding the same ligand, similar to the
different collagen-binding modes of the von Willebrand factor–A3
domain and its structural homologue the integrin ␣2 I-domain.42,43
However, the observed partial conservation of the LAIR-1 collagenbinding site in GPVI, in combination with the absence of conservation of the proposed GPVI binding site in GPVI orthologues, is
suggestive of similar collagen-binding modes for LAIR-1 and
GPVI and warrants further studies into the GPVI-collagen
interaction.
Libraries of overlapping synthetic triple-helical peptides encompassing the complete collagen II and III triple-helical domain have
been instrumental in the identification of recognition sites for a
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BLOOD, 18 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 7
A
STRUCTURE AND COLLAGEN-BINDING SITE OF LAIR-1
1371
B
180˚
C
180˚
D
E
180˚
Figure 5. Conservation of putative collagen-binding residues in LAIR-1 and GPVI. (Left) Surface representations of the LAIR1-CBD (A) and the GPVI ectodomain (D)
showing in blue residues that contribute to collagen binding according to mutagenesis data, in red residues that form the LAIR-1 collagen binding surface based on the NMR
titrations, and in green residues that contact docked collagen peptides in GPVI. Data for LAIR-1 and GPVI are from this study and Horii et al,14 respectively. GPVI residues, L53
and F54 that are part of the docking interface as well as the equivalent of the LAIR-1/collagen interface, are shown in green. (Right) Surface representations in 2 orientations of
LAIR-1 (B-C) and GPVI (E) showing in red residues that are strictly conserved within the LAIR-family (B), within both the LAIR and GPVI families (C) and, residues conserved
within the GPVI family only (E). Note that putative collagen-binding residues of LAIR-1 are significantly conserved in both LAIR and GPVI families, whereas putative
collagen-binding residues of GPVI are not at all conserved. Conservation is based on the alignments shown in supplemental Figures 5 and 6. The orientation of LAIR is identical
to Figure 2B, whereas GPVI is depicted with its D1 domain in an orientation similar to LAIR-1.
range of collagen-binding proteins, including LAIR-1, GPVI, von
Willebrand factor, SPARC, and integrin ␣2␤1.7,12,26,44-46 Biophysical techniques such as polarimetry confirm the triple-helical nature
of these peptides,28 whereas crystal structures, albeit of shorter
synthetic peptides, show that they display the proper register with a
1-residue stagger between the 3 peptide strands.47-50 LAIR-1 was
found to bind at least 6 peptides of the collagen III library.7 The
existence of multiple binding sites on collagen is also evident from
BIAcore data showing that on average approximately 10 LAIR-1
binding sites are present per collagen II or III triple helix.6 Our data
reveal some intriguing differences between the binding of LAIR-1
mutants to whole collagen III and to collagen peptide III-30.
Mutants E61A and R65A show a strong reduction in binding to
collagen III, whereas binding to peptide III-30 is not affected for
mutant R65A and only slightly reduced for mutant E61A (supplemental Figure 4). Other mutations, such as R59A and Q111A,
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BLOOD, 18 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 7
BRONDIJK et al
reduce binding to peptide and collagen to similar extents. The
observed differences between our peptide- and collagen-binding
studies could arise from several factors including subtle differences
in the conformation of the triple helix. Crystal structures of
synthetic triple-helical peptides show a 7/2 helical twist in imino
acid–rich regions going toward 10/3 or intermediate twist in the
more amino acid–rich collagen regions.47-50 To date, there is no
high-resolution collagen structure available and there is no consensus model for the collagen helical twist; both a 7/2 and a 10/3
helical twist of collagen have been proposed.51,52 Another factor
that might be responsible for the observed differences between
peptide and collagen binding is LAIR-1 contacting more than one
triple-helix in fibrillar collagen simultaneously, a situation that
cannot be reproduced in the model peptide. A similar situation
exists for integrin ␣2␤1 where the context of one specific binding
site is important to determine binding affinity,28 suggesting adjacent residues in the collagen fiber may affect binding. Alternatively,
the superstructure of the collagen microfibril could result in
variable exposure of different segments of the collagen triple helix
on the surface of the fiber53; some LAIR-1 binding sites present in
collagen peptides may therefore not be exposed at the surface of a
collagen fibril and these would be inaccessible for LAIR-1 binding.
This situation could lead to different effects of mutations on
binding to a collagen peptide and intact fibrous collagen. Thus,
synthetic triple-helical peptides are invaluable tools for the identification of collagen-protein interaction sites, but at least in the case
of LAIR-1, results obtained with these peptides will need to be
validated in the context of whole collagen.
In summary, we have clearly defined a surface on LAIR-1 that is
involved in the interaction with collagen and this surface is
conserved across species. We have identified key binding-site
residues that are important in the interaction of LAIR-1 with
collagen. Although the LAIR-1 collagen-binding site is well
conserved, there are some surprising differences between species.
The LAIR-1 collagen-binding site is remarkably different from the
proposed collagen-binding site of the LAIR-1 homologue, platelet
protein GPVI. However, our analyses suggest that with the
presently available data it cannot be excluded that GPVI binds
collagen at a site similar to that of LAIR-1 suggesting a common
structural basis for the recognition of collagen by both proteins.
Further studies are needed into the GPVI-collagen interaction to
clarify this issue. The available crystal structures will facilitate the
development of compounds targeted at specifically modulating
immune responses via the LAIR-1/collagen interaction or thrombosis through GPVI.
Acknowledgments
We thank the European Synchrotron Radiation Facility for providing data collection facilities and the beamline scientists at ID23-1
for their help with data collection. We thank Dr Alexandre Bonvin
for his help and advice with regard to the docking experiments.
This work was supported by the Netherlands Heart Foundation
(NHS2005B238), the Netherlands Foundation for Chemical Research NWO/CW, the Center for Biomedical Genetics, and the EU
FP6 project EU-NMR (contract no. RII3-026145).
Authorship
Contribution: R.J.L., T.R., and L.M. initiated the work and
performed the mutagenesis studies; T.H.C.B. and J.B. purified
proteins and did the crystallographic studies; J.B., H.I., H.W., and
R.B. performed and analyzed the NMR experiments; R.W.F.
provided the synthetic peptide; T.H.C.B. performed the docking;
and T.H.C.B., L.M., and E.G.H. designed the experiments and
wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Eric G. Huizinga, Crystal and Structural
Chemistry, Bijvoet Center for Biomolecular Research, Utrecht
University, Padualaan 8, Utrecht 3584 CH, The Netherlands;
e-mail: [email protected].
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2010 115: 1364-1373
doi:10.1182/blood-2009-10-246322 originally published
online December 10, 2009
Crystal structure and collagen-binding site of immune inhibitory
receptor LAIR-1: unexpected implications for collagen binding by
platelet receptor GPVI
T. Harma C. Brondijk, Talitha de Ruiter, Joost Ballering, Hans Wienk, Robert Jan Lebbink, Hugo van
Ingen, Rolf Boelens, Richard W. Farndale, Linde Meyaard and Eric G. Huizinga
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