From the § Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Center and Institute of Biomembranes, HP G03.647, P. O. Box 85500, 3508 GA Utrecht, The Netherlands and the ¶ Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Received for publication, July 21, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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Von Willebrand factor (vWF) is a multimeric
glycoprotein that mediates platelet adhesion and thrombus formation at
sites of vascular injury. vWF functions as a molecular bridge between
collagen and platelet receptor glycoprotein Ib. The major
collagen-binding site of vWF is contained within the A3 domain, but its
precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment
of antibody RU5 that inhibits collagen binding. The structure shows
that RU5 recognizes a nonlinear epitope consisting of residues 962-966, 981-997, and 1022-1026. Alanine mutants were constructed of
residues Arg963, Glu987,
His990, Arg1016, and
His1023, located in or close to the epitope. Mutants
were expressed as fully processed multimeric vWF. Mutation of
His1023 abolished collagen binding, whereas mutation of
Arg963 and Arg1016 reduced collagen binding by
25-35%. These residues are part of loops Platelet adhesion to damaged vessel walls is the first step in the
formation of an occluding platelet plug, which leads to the arrest of
bleeding during normal hemostasis. Platelet adhesion can also cause
thrombotic complications such as the occlusion of atherosclerotic
arteries (1). The multimeric glycoprotein von Willebrand factor
(vWF)1 plays an essential
role in platelet adhesion under conditions of high shear stress (2, 3).
In this process vWF serves as a molecular bridge that links collagen
exposed by the damaged vessel wall to glycoprotein Ib located on the
platelet surface. Collagens that act as binding sites for vWF include
types I and III in perivascular connective tissue and type VI in the
subendothelial matrix (3, 4).
Mature vWF consists of a 2050-residue monomer that contains multiple
copies of so-called A, B, C, and D type domains and one CK
(cystine knot) domain arranged in the order
D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (1, 3). Disulfide bond formation
between N-terminal D3 domains and between C-terminal CK domains
generates vWF multimers that consist of up to 80 monomers. The A1
domain contains the binding site for glycoprotein Ib (5). The A3 domain
(residues 920-1111) contains the major binding site for collagen types
I and III (6). The multimeric structure of vWF is essential for high
affinity collagen binding (7). Multimeric vWF binds collagen with an apparent Kd of 1-7 nM (8), while a
recombinant A3 domain has a much higher Kd of 2 µM (9). Deletion of the A2 and D4 domains, which flank
the A3 domain, or deletion of the A1 domain do not decrease collagen
binding of multimeric vWF (6, 8). These data show that a monomeric A3
domain contains a fully active collagen-binding site, the only
requirement for tight binding to collagen being the presence of
multiple A3 domains within one vWF multimer.
Integrin I-type domains are homologous to vWF A-type domains (10, 11).
I domains of integrin The location of the collagen-binding site in the vWF-A3 domain is not
known. Crystal structures of A3 do not display a collagen-binding groove in the top face, instead, the surface of A3 is rather smooth (15, 16). Although the MIDAS motif is partly conserved, binding of A3
to collagen does not require a metal ion (17, 18), and no metal ion is
observed in crystal structures of A3. Moreover, point mutations in the
MIDAS motif of A3 do not disrupt collagen binding (8, 16) showing that
the motif is not involved in collagen binding, at all. Site-directed
mutagenesis studies of other residues in the top face of A3 have
yielded conflicting results. Cruz et al. (19) reported in
abstract form that amino acid substitutions D1069R, R1074D, R1090D, and
E1092R resulted in a 50% reduction in binding of monomeric A3 to
collagen. Van der Plas et al. (8), however, observed normal
collagen binding of fully processed multimeric vWF containing mutations
D1069R, D1069A, or R1074A. In the same study, mutations
V1040A/V1042A, D1046A, and D1066A also displayed normal collagen
binding, suggesting that the collagen-binding site of vWF-A3 is not
located in its top face.
The crystallographic study presented here was conducted to provide new
clues on the location of the collagen-binding site of the vWF-A3
domain. We determined the structure of the A3 domain in complex with a
Fab fragment of monoclonal antibody RU5, which inhibits binding of vWF
to collagen. Site-directed mutagenesis of residues located in the
epitope region show that the collagen-binding site is located distant
from the top face of A3.
Purification of vWF-A3 and RU5--
Recombinant selenomethionine
(Se-Met) A3, comprising residues 920-1111 of human vWF, was expressed
and purified as described before (15). For production of monoclonal
antibody RU5 (IgG2a, Determination of RU5-variable Domain Sequences--
The amino
acid sequence of the variable domain of the heavy chain
(VH) of RU5 was deduced from a cDNA nucleotide
sequence. Cloning and sequencing was carried out using established
procedures. Total RNA was extracted from 4 × 106 RU5
hybridoma cells with RNAzol using the RNA isolation protocol of the
supplier (Campro Scientific, Veenendaal, The Netherlands). First-strand
cDNA was synthesized using Superscript II reverse transcriptase
(Life Technologies, Inc., Rockville, MD) in the presence of 3.2 units/µl RNase inhibitor RNaseOUT (Life Technologies, Inc.).
cDNA, encoding the VH domain, was amplified by
polymerase chain reaction with Pwo DNA polymerase (Roche Molecular
Biochemicals, Mannheim, Germany). A backward variable region consensus
primer (B4: CCA GGG GCC AGT GGA TAG ACA AGC TTG GGT GTC GTT TT) was
used together with a forward primer (B3c: CGG ATG GCC AGG T(C/G)(A/C) AGC TGC AG(C/G) AGT C(A/T)G G) that hybridizes with a consensus sequence in the JH region (21). The polymerase chain
reaction product was extended with a 3'-A overhang using Taq
DNA polymerase (Promega, Madison, WI) and cloned into the pCR2.1-TOPO
vector according to the manufacturer's protocol (Invitrogen, Leek, The Netherlands). Nucleotide sequences of three clones were determined (GenBankTM accession number AF286587) (Fig. 1A).
For determination of the sequence of the variable domain
(VL) of the RU5 Crystallization and Data Collection--
Crystals of the
(Se-Met) A3·RU5 complex were grown by hanging-drop vapor diffusion at
4 °C using a protein concentration of 15 mg/ml and a precipitant
solution consisting of 13% (v/v) isopropanol, 22% (v/v)
2-methyl-2,4-pentanediol, and 100 mM cacodylic acid, pH
5.3. The crystal used for structure determination had a size of
0.1 × 0.1 × 0.2 mm3 and was equilibrated in a
cryoprotectant solution consisting of 8% isopropanol, 30%
2-methyl-2,4-pentanediol, 100 mM cacodylic acid, pH 5.3. X-ray diffraction data were collected on beam line BW7B of the
synchrotron radiation facility of the EMBL outstation (Hamburg,
Germany) with a Mar345 imaging plate (Mar, Evanston, IL). Data
reduction, merging, and scaling were performed with DENZO and SCALEPACK
(23). Diffraction data statistics can be found in Table I.
Diffraction of the A3·RU5 crystal was anisotropic. For structure
determination, anisotropic B-factors were applied with SCALEIT and
SFTOOLS (24) to correct for the observed anisotropy.
Structure Determination and Refinement--
A self-rotation
function calculated with POLARRFN (24) and the unit-cell volume
suggested the presence of three A3·RU5 complexes in the asymmetric
unit (a.u.) with a VM of 3.3 Å3/Da and a
solvent content of 63%. Initial attempts to solve the structure by
molecular replacement failed. Therefore, a rather weak anomalous signal
(
For model building, sequences of the constant domain of the light chain
(CL) and of the constant domain of the heavy chain (CH1) of RU5 were taken from IgG2a, Construction of vWF Point Mutants--
Point mutations were
introduced in the vWF-A3 domain using the QuikChange method
(Stratagene, La Jolla, CA) and specific primers (Amersham Pharmacia
Biotech, Roosendaal, The Netherlands). First, a 518-base pair
NheI-Csp45I fragment corresponding to
amino acid residues 940-1113 of mature vWF was subcloned into
pBluescript SKII (Stratagene, La Jolla, CA). To this end a unique
Csp45I restriction site was introduced at position 5628 of
expression vector pNUT-vWFcas (8) in the following way. The
BamHI-EcoRV fragment of pNUT-vWFcas was ligated
into pBluescript SKII. The Csp45I site was introduced using
QuikChange and confirmed by sequencing. The
NheI-EcoRV fragment containing the new
Csp45I site was ligated into pNUT-vWFcas generating pNUT-vWFcas2. Next, the NheI-Csp45I fragment of
pNUT-vWFcas2 was made blunt by filling in the 5'-overhangs using Pwo
DNA polymerase. Ligation of this fragment into
EcoRI-AccI-digested and Pwo-treated pBluescript
SKII, produced mutagenesis plasmid pBSvWF-NC. This plasmid retains the
NheI and Csp45I restriction sites. Point
mutations R963A, E987A, H990A, R1016A, and H1023A were introduced in
pBSvWF-NC. Mutation H1023A causes the disappearance of a
NsiI restriction site. The
NheI-Csp45I fragment of this mutant was ligated
directly into pNUT-vWFcas2 and confirmed by restriction analysis. The
NheI-Csp45I fragment of the other point mutants
were ligated into pNUT-vWFcas2-H1023A, with reappearance of the
NsiI site.
Expression, Purification, and Characterization of vWF--
vWF
was stably expressed in baby hamster kidney cells overexpressing furin,
necessary for proper removal of vWF propeptide (6, 34). vWF was
purified by immunoaffinity chromatography using monoclonal antibody
RU8, which is directed against the D4 domain (6). vWF-containing
fractions were pooled and stored in aliquots at
The concentration of vWF was determined by a sandwich enzyme-linked
immunosorbent assay using polyclonal
The multimeric structure of vWF was analyzed by agarose gel
electrophoresis followed by Western blotting as described by Lawrie et al. (35).
Binding of vWF to monoclonal antibody RU5 was analyzed as follows.
Microtiter-plate wells (Costar, Cambridge, MA) were coated with 2.5 µg/ml polyclonal Collagen Binding Assay--
vWF binding to fibrillar human
placenta collagen type I (Sigma Chemical Co., St. Louis. MO, cat. no.
C-7774) and collagen type III (Sigma, cat. no. C-4407) was studied in a
solid-state binding assay according to Van der Plas et al.
(8). Collagen was coated at 50 µg/ml instead of 100 µg/ml as used
previously (8). A vWF concentration of 2.5 µg/ml was used in the
binding experiments.
A3·RU5 Structure Determination and Refinement--
The structure
of the vWF (Se-Met) A3 domain in complex with an Fab fragment of RU5
was solved to 2.0-Å resolution. The structure was determined using the
anomalous signal from selenium atoms in (Se-Met) A3 to position two A3
molecules and was completed by molecular replacement. The a.u. contains
three A3·Fab complexes. The structure has been refined to an
R-factor of 22.7% and a free R-factor of 26.4%
(Table I).
The sequence of the RU5 VL domain could not be determined
from cDNA and was therefore deduced from electron density aided by
an alignment of 110 Fab VL sequences. The identity of 13 amino acid residues could not be determined uniquely (Fig.
1). The structure displays good model
geometry with 88.8% of the residues in the most favored region of the
Ramachandran plot and 10.8% in the additionally allowed region.
Residue Thr51 of RU5 VL domains occurs in the
disallowed region of the Ramachandran plot, but its electron density is
convincing.
The a.u. consists of three A3·RU5 complexes that are denoted A, B,
and C. The overall structure of the (Se-Met) A3 molecule is the same as
the structure of native A3 (15, 16). It consists of a central
six-stranded Crystal Packing and Differences between Complexes--
In the a.u.
A3·RU5 complexes B and C form a tightly interacting anti-parallel
dimer and are related by 2-fold n.c.s. A3·RU5 complex A forms a
similar anti-parallel dimer with a crystallographically related complex
A'. Complexes within dimers B-C and A-A' have large contact areas of
932 Å2 and 1454 Å2, respectively (GRASP
(37)). These large interaction surface areas suggest that the dimer
of A3·RU5 complexes may also be stable in solution. Dynamic
light-scattering measurements, however, clearly indicated that
the A3·RU5 complex does not form dimers in solution. Therefore,
dimers observed in the crystal are a result of crystal packing.
Superposition of C The Epitope of RU5--
A3 interacts with RU5 through a nonlinear
epitope comprising residues in loop Conformational Changes in A3--
To analyze whether binding of
RU5 causes conformational changes in A3, we compared models of A3 in
the A3·RU5 complex with two structures of free
A32 (15, 16). The two
structures of free A3 were determined from different crystal forms with
unrelated crystal-packing interactions. In both crystal forms, two
molecules are present in the a.u. Because the A3·RU5 complex was
obtained with (Se-Met) A3, we also included the structure of free
(Se-Met) A3 in the comparison to detect possible structural differences
caused by Se-Met.3 Root mean
square coordinate differences after pairwise superimposing C
The three conformational diverse loops located in the RU5 epitope are
Distant from the RU5 epitope conformational variation is observed for
loops
Loop Selection and Characterization of vWF Mutants--
To confirm the
location of the collagen-binding site indicated by the A3·RU5
complex, we constructed charged-to-alanine mutations of five residues.
These residues are located within 5 Å of the RU5 Fab fragment and are
solvent-exposed in free A3. Residues selected were Arg963
located in loop Collagen Binding of vWF Mutants--
The effect of point mutations
on vWF binding to collagen types I and III was investigated by
enzyme-linked immunosorbent assay (Fig.
6). Similar results were obtained for
both types of collagen. Mutation H1023A almost completely abolished
collagen binding. The level of residual binding observed for this
mutant was similar to binding observed for The current study was aimed at locating the collagen-binding site
of the vWF-A3 domain. For this purpose we solved the crystal structure
of A3 in complex with a Fab fragment of RU5 that inhibits collagen
binding. The structure of the complex shows that RU5 binds to residues
within A3 sequences 962-966, 981-997, and 1022-1026. These residues
are located in To confirm the location of the collagen-binding site, we constructed
five charged-to-alanine mutations of residues located in or close to
the RU5 epitope. The multimer distribution of these mutants was similar
to that of wild-type vWF. Therefore, observed differences in collagen
binding are not caused by the known dependence of collagen binding on
vWF multimer size (7). All five mutants bound normally to RU5, which
shows that none of the mutated residues plays a dominant role in the
A3·RU5 interaction and, more importantly, that the conformation of A3
in the neighborhood of the epitope and the collagen-binding site is not disturbed.
Mutation H1023A abolished collagen binding almost completely, residual
binding being similar to that observed for 3
4 and
1
2 and
-helix 3, respectively, and lie near the bottom face of the domain.
His1023 and flanking residues display multiple
conformations in available A3-crystal structures, suggesting that
binding of A3 to collagen involves an induced-fit mechanism. The
collagen-binding site of A3 is located distant from the top face of the
domain where collagen-binding sites are found in homologous integrin I domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chains
1,
2,
10, and
11 all possess
collagen-binding sites. A crystal structure of the
2-I
domain reveals binding of a collagen-like peptide to a groove in the
surface of the "top" face of the domain (12). This groove contains
a so-called metal ion-dependent adhesion site (MIDAS) (13,
14), which engages a glutamate residue of collagen.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) hybridoma cells were injected in
mice, and ascites fluid was collected (Eurogentec, Seraing, Belgium).
IgG was purified on a protein G-Sepharose column, and Fab fragments
were generated using an ImmunoPure Fab kit (Pierce, Rockford, IL).
RU5-Fab was further purified with A3-affinity chromatography.
For that goal, 10 mg of A3 was irreversibly bound via its N-terminal
histidine tag to cobalt(III)-iminodiacetate-chelating Sepharose
(Amersham Pharmacia Biotech, Uppsala, Sweden), according to a procedure
described by Hale (20). The RU5-Fab fragment was loaded onto the
A3-affinity column, washed with PBS, and subsequently eluted with 50 mM triethylamine solution (pH 10.0). Some aggregates were
removed on a Superdex 75 HR 10/30 gel filtration column (Amersham
Pharmacia Biotech). Running buffer was 10 mM Tris-HCl, pH
8.0, 25 mM NaCl. Next, RU5-Fab was mixed with a 2-fold
molar excess of (Se-Met) A3. The A3·RU5 complex was separated from
excess A3 by gel filtration chromatography. Dynamic light scattering
measurements on a Dynapro-801 DLS instrument (Protein Solutions,
Charlottesville, VA) indicated the presence of 69-kDa particles in
agreement with an expected molecular mass of about 72 kDa of the
complex between A3 and RU5.
-light chain, the same procedure was
used as for the VH domain. For cloning of VL
cDNA, one backward variable region consensus primer (PD1: GAT ATT GTG
ATG ACC CAG TCT (C/G)T) and two forward variable-region consensus
primers (PD3: CAG GAA ACA GCT ATG ACC GAG CTC GTG ATC ACC CAG TCT CC;
PD4: TGT AAA ACG ACG GCC AGT TCT AGA TGG TGG GAA GAT GGA) were used.
However, determination of the sequence was hampered by the abundance of
mRNA encoding the light chain of the nonproducing myeloma fusion
partner of the RU5 hybridoma cell, a known drawback of myeloma cell
line P363Ag8.653 (22).
Fano/
(F)
= 1.18) arising
from the presence of Se-Met in A3 was used to locate Se sites from an
anomalous Patterson map. Six Se sites could be assigned to
Met947, Met998, and Met1097 of two
A3 molecules on the basis of inter-atomic distances and the observed
2-fold non-crystallographic symmetry (n.c.s). The positioning of two A3
molecules in the a.u. allowed further molecular replacement to proceed
in a straightforward manner. A Fab fragment of protein data bank entry
2MPA (25, 26) was oriented using the program AmoRe (27) followed by
Patterson correlation refinement using the program CNS (28, 29).
Cross-translation functions calculated with CNS identified the position
of three Fab fragments. A third A3 molecule was placed on the basis of
n.c.s. The a.u. finally contained three A3·RU5 complexes with an
R-factor of 42.6% after rigid body refinement.
monoclonal antibody 4-4-20 (30, 31). The sequence of residues 1-110 of
the VL domain of RU5 (Fig. 1B) was derived from
the electron density aided by a consensus sequence based on an
alignment of 110 Fab sequences. For model refinement, cycles of
rebuilding using O (32) and positional and B-factor refinement using
CNS were performed until convergence. Cross-validation was used
throughout refinement using a 5% test set of reflections. Refinement
used the maximum-likelihood algorithm (33). Bulk solvent correction and
anisotropic scaling of diffraction data were applied. During the first
cycles of refinement n.c.s. restraints were used. Based on the behavior
of the free R-factor, n.c.s. restraints were omitted in
later stages of refinement. Water molecules were placed in difference
electron density peaks with a peak height of at least 2.8
, a
distance of 2.5-3.4 Å to a hydrogen bond donor or acceptor, and a
B-factor smaller than 65 Å2.
20 °C until use.
-vWF and horseradish peroxidase-conjugated polyclonal
-vWF (Dako, Glostrup, Denmark) for
immobilization and detection, respectively (6). Normal pooled plasma
from 40 healthy donors was used as a reference.
-D'D3 (8) in 50 mM carbonate buffer, pH 9.6 (3 h, 20 °C). Wells were washed with PBS/0.1% Tween-20 (PBS/T) and blocked with 3% BSA in PBS/T (1 h, 37 °C). Wells were incubated with 100 ng/ml vWF in PBS/T, including 3% BSA (1 h, 37 °C). After washing, 5 µg/ml RU5 was added (1 h, 37 °C).
Wells were washed and incubated with horseradish peroxidase-conjugated rabbit anti-mouse antibody (Dako) diluted 1:2500 in PBS/T containing 3% BSA (1 h, 37 °C). O-Phenylenediamine was used as
substrate for detection.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Diffraction-data and refinement statistics
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Fig. 1.
Amino acid sequences of RU5 VL
and VH domains. CDRs (underlined) were
defined according to Kabat et al. (23). Strict sequential
numbering (#) is used throughout the text. Numbering according to the
convention of Kabat et al. (23) (#K) is also
shown. A, the amino acid sequence of VH was
deduced from cDNA. Residues 1-7 could not be deduced precisely due
to the use of a primer complementary to the 5'-coding region.
Asn56 in CDRH2 (shaded) is
N-glycosylated. The nucleotide sequence of VH
has been deposited in the GenBankTM data base under accession number
AF286587. B, the amino acid sequence of the VL
domain was deduced from the electron density of the A3·RU5 complex
and an alignment of 110 Fab VL chains. Side chains of 13 amino acid residues that could not be determined unambiguously from the
electron density and the consensus sequence are shown as
lowercase characters.
-sheet on both sides flanked by
-helices. The final
model comprises amino acid residues 921-1110 of A3 molecules in
complexes A and B and residues 920-1110 of the A3 molecule in complex
C. Positions of the N and C termini of A3, including disulfide bond
Cys923-Cys1109 that connects these termini, are
poorly defined. The model of RU5 consists of residues 1-211 of the
light chains and residues 1-129 and 136-216 of the heavy chains.
Residues 130-135 of the heavy chains display very weak electron
density and are excluded from the model. Disorder of this loop is a
commonly observed feature in Fab structures (36). Asn56 of
the VH domain is N-glycosylated. Electron
density near Asn56 accounted for a GlcNAc(-Fuc)-GlcNAc
moiety in complexes A and B and only a GlcNAc-Fuc moiety in complex C. The model contains one cacodylate ion from the crystallization solution.
atoms of the three A3·RU5 complexes
gives large root mean square coordinate differences ranging from 0.7 to
0.9 Å. When superpositions are limited to A3 molecules with bound
VH and VL domains, root mean square coordinate
differences of 0.5-0.6 Å are obtained. Visual inspection of these
superpositions (Fig. 2) shows that the
A3·RU5 interaction region is well conserved, whereas regions of A3
and the variable domains located further away from the interaction
region superimpose less well. A more detailed inspection of the
A3·RU5 binding region reveals that all side-chain conformations are
very similar (data not shown). In conclusion, the A3·RU5 interaction
region is well conserved among the three complexes in the a.u.
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Fig. 2.
Structural overlay of
C traces of the three A3·RU5 Fab
complexes in the asymmetric unit. For calculation of the best
superposition, C
atoms of A3 and variable domains of
RU5-Fab were used. The A3·RU5 interaction region is well conserved
among the three complexes, but relatively large differences are shown
in orientations of the RU5 constant domains with respect to its
variable domains and A3. The diagram was generated with MOLSCRIPT
(43).
1
2, loop
3
2 followed by
helix
2, and loop
3
4 (Table II).
All three loops that contribute to the epitope are located in
the bottom face of A3 (Fig. 3). The N and
C termini of A3 that are also located in the bottom face do not
interact with RU5. This observation is in agreement with the fact that RU5 was raised against complete vWF in which the termini connect A3 to
flanking A2 and D4 domains. Residues of RU5 that interact with loops
1
2 and
3
4 are located in CDRL1 and
CDRL2 (Fig. 1). All six CDRs interact with residues in the
contiguous segment formed by loop
3
2 and helix
2. The A3·RU5
interactions involve five hydrogen bonds and one salt bridge. The
buried surface area of A3 molecules in the complex is ~1200
Å2, which is about 7% of the total surface area of A3.
Interestingly, the fucose moiety attached to residue Asn56
in CDRH2 of complex A interacts with
Lys988-Ala989 of A3. The carbohydrate moieties
attached to Asn56 in complexes B and C do not interact with
A3.
The epitope of RU5 and loops of A3 that show conformational variation
among eight models of A3 from four different crystal structures
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Fig. 3.
Ribbon diagram of A3 and RU5 variable
domains. Residues of A3 that are part of the epitope of RU5
are indicated by yellow spheres. Regions of RU5 CDRs that
interact with A3 are color-coded in light blue.
Regions of A3 that show different conformations among four crystal
structures are shown in purple trace. Disulfide bond
Cys923-Cys1109 is shown in green
ball-and-stick. The -helices and
-strands of A3 are depicted
in blue and red, respectively. For clarity,
-helix 2 is not shown by a ribbon but in
"coil" representation. The VH domain of RU5
is in brown; the VL domain is in
green. The diagram was generated with MOLSCRIPT (43) and
RASTER3D (44).
atoms of all eight models range from 0.24 to 0.81 Å.
Large differences are restricted to three loops located within the
RU5-binding site and to two loops that are located distant from the
epitope (Fig. 3 and Table II).
1
2,
3
2, and
3
4 (Fig.
4A). Loop
1
2 has a
unique conformation in the A3·RU5 complex, indicating that this
conformation is induced by RU5. Loops
3
2 and
3
4 have
considerable conformational freedom. Conformations observed in the
A3·RU5 complex are, however, not systematically different from the
conformations observed in free A3, indicating that the conformations
observed in the complex are not induced by RU5.
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Fig. 4.
The conformation of five loops varies among
different crystal structures of A3. Models compared include free
A3,2 free (Se-Met) A3, and A3 from the A3·RU5 complex.
A, stereo view of loops 1
2 (residues 961-963),
3
2 (983), and
3
4 (1019-1025). Residues of A3 in
A3·RU5 complex A are shown with the thick line. A3 domain
A from the structure of Huizinga et al. (15) is shown in
gray. B, stereo diagram of residues
Ser938-Phe939-Pro940 in the top
face of A3. Buried and exposed conformations of Phe939 are
labeled. C, stereo diagram of loop
5
6 (residues
1078-1083). The aberrant A3 domain B from the structure of Bienkowska
et al. (16) is shown with the thick line. The
diagram was generated with MOLSCRIPT (43).
1
1 and
5
6. Loop
1
1 is located in the top face
of the domain and contains part of the vestigial MIDAS motif. Multiple
conformations are observed for residues
Ser938-Phe939-Pro940 (Fig.
4B). Phe939 is solvent-exposed in A3·RU5
complex C and all models of free A3. In complexes A and B
Phe939 is buried in the hydrophobic core of A3. The buried
conformation is likely caused by crystal packing interactions involving
A3 residues 940-942. These packing interactions would not allow for the position of Pro940 observed in models of A3 that have
an exposed conformation of Phe939. Because both the exposed
and buried conformations of Phe939 are observed in the
three A3·RU5 complexes, the conformation of this loop is certainly
not determined by binding of RU5.
5
6 located in a side face of A3 has an aberrant conformation
in A3 domain B of the structure of Bienkowska et al. (16) (Fig. 4C). This aberrant conformation coincides with crystal
contacts unique to molecule B. No crystal contacts are
present in the other structures. The conformations observed in the
A3·RU5 complex are not systematically different from the
conformations observed in free A3. Thus, binding of RU5 does not cause
conformational changes in regions of A3 that are distant from the epitope.
1
2, Glu987 (
3
2),
His990 (
2), Arg1016 (
3), and
His1023 (
3
4). Mutants were produced as multimeric vWF
in stable baby hamster kidney cell lines. The multimer distribution of
vWF mutants and wild-type vWF, as analyzed by agarose gel
electrophoresis, were indistinguishable (data not shown). RU5 binding
to purified point mutants was not significantly different from RU5
binding to wild-type vWF (Fig. 5).
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Fig. 5.
Binding of monoclonal antibody RU5 to vWF
mutants. vWF immobilized in microtiter plate wells via polyclonal
D'D3 was incubated with 5 µg/ml RU5. Bound RU5 was detected by
horseradish peroxidase-conjugated rabbit-anti-mouse antibody as
described under "Experimental Procedures." Wild-type vWF and
A3-vWF, which lacks the A3 domain (6), were used as positive and
negative controls, respectively. Binding of RU5 to vWF mutants is shown
as a percentage of wt-vWF. Each data point represents the
mean ± S.D. of two measurements in duplicate.
A3-vWF, a deletion mutant
that lacks the entire A3 domain (6). Collagen binding of mutants R963A and R1016A was reduced by 25-35%, whereas collagen binding of mutants
E987A and H990A was normal.
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Fig. 6.
Binding of vWF point mutants to collagen
types I and III. Collagen type I (white bar) or III
(black bar) was coated in microtiter-plate wells. Wells were
incubated with vWF at a concentration of 2.5 µg/ml, and bound vWF was
detected as described under "Experimental Procedures." A3-vWF
was used as a negative control. Bound vWF mutant is shown as a
percentage of wt-vWF. Each data point represents the mean ± S.D.
of three independent experiments performed in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix 2 and in loops
1
2,
3
2, and
3
4 at the bottom of one of the side faces of the A3 domain (see
Fig. 3). Comparison of structures of A3 shows that RU5 binding does not
induce long range conformational changes. This excludes a mechanism in
which RU5-induced conformational changes inhibit collagen binding. It
seems likely, therefore, that RU5 inhibits collagen binding by steric
hindrance, which implies that the collagen-binding site is located at
or close to the RU5 epitope.
A3-vWF, a deletion mutant
that lacks the entire A3 domain. Therefore, His1023 plays a
central role in A3-mediated collagen binding. His1023 is
located in loop
3
4 and lies at the edge between the "front" face of the domain, formed by helices
2 and
3 and strand
3, and the bottom face, which is composed of several loops and contains the N and C termini (Fig. 7). A small
reduction of collagen binding was observed for mutants R963A and R1016A
located in the bottom and front face of the domain, respectively.
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Fig. 7.
Residues of vWF-A3 involved in collagen
binding. Stereographic representation of the vWF-A3 domain showing
amino acid residues that have been mutated to alanine in
ball-and-stick. Mutation of His1023
(magenta) abolishes collagen binding almost completely.
Mutation of Arg963 and Arg1016
(green) reduces collagen binding by 25-35%. Mutation of
Glu987 and His990 (gray) does not
have an effect on collagen binding. To illustrate the range of
conformations of His1023 observed in different crystal
forms of A3, two conformations of this residue and the backbone trace
of flanking residues are shown. Residues that have been mutated in
previous studies (8, 16, 19) are located in the top face of A3 and are
indicated by black spheres. The diagram was generated with
MOLSCRIPT (43) and RASTER3D (44).
Interestingly, His1023 and flanking residues display a
large variety of conformations among eight models of A3 (Figs. 4 and
7). In some A3 structures His1023 protrudes prominently
from the surface of the domain, which may be a favorable position for
interaction with collagen. Multiple conformations are also observed for
loop 3
2. Like His1023, loop
3
2 is located at
the edge between the front and bottom faces of A3. Because we did not
mutate residues in loop
3
2, its involvement in collagen binding
remains to be established. The observed flexibility of
His1023 suggests that collagen binding may involve an
induced-fit mechanism in which significant conformational changes occur
in loop
3
4 upon binding of A3 to collagen.
The amino acid sequence of collagen that is recognized by vWF-A3 has not yet been identified. We hypothesized previously that negatively charged residues in A3 could interact with basic residues on collagen (15). Residues now implicated in collagen binding are positively charged. Therefore, interaction of A3 with negatively charged residues on collagen appears more likely.
In contrast to binding sites of other collagen binding domains, like
the 1 and
2-I domains and the A domain of
Staphylococcus aureus adhesin (12, 38), the
collagen-binding region of A3 does not have a groove or trench that
could accommodate a collagen triple helix. The front face of the
domain, harboring Arg1016, is rather flat. The bottom face,
which contains Arg963 is less smooth, but no groove is
present. Docking of a collagen triple helix on the A3 domain is not
straightforward. In particular, it is not obvious how
His1023, Arg963, and Arg1016 could
simultaneously contact a triple helix in an extended conformation. To
define the collagen-binding site more precisely, characterization of
additional mutants will be necessary.
Previously, the collagen-binding site of A3 was proposed to be located
at its top face (15, 19) similar to the homologous I domains of
integrins 1
1 and
2
1 (12, 39-41). Point mutations introduced in the top face of an A3 monomer (19) and in multimeric vWF
(8) gave conflicting results. Our results now show conclusively that
the collagen-binding site is located close to the bottom face and not
in the top face of A3.
Although our results rule out a role for the top face of the A3 domain in collagen binding, this side of the molecule may still be engaged in other interactions, such as binding of the A1 domain. Interaction between A1 and A3 has been suggested to play a role in activation of the A1 domain for binding to platelet receptor glycoprotein Ib (42). Interesting in this respect are the buried and solvent-exposed conformation observed for residue Phe939, which is located close to the vestigial MIDAS motif in the top face of the domain (Fig. 4B). Solvent exposure of Phe939 has been proposed to stabilize the buried Asp934 of the vestigial MIDAS motif in the absence of a bound metal ion (16). Our observation of a buried conformation shows that exposure of Phe939 is not critical for structural stability. The two conformations of Phe939 may, however, be relevant for the putative interaction between A1 and A3, because the shape and hydrophobicity of the upper surface of A3 differs significantly between the solvent-exposed and buried conformation.
In conclusion, the collagen-binding site of vWF-A3 is distinctly
different from collagen-binding sites of I domains of integrins 1
1 and
2
1.
vWF-A3 residues involved in collagen binding are located close to the
bottom face of the domain. His1023 is essential for
collagen binding, whereas Arg963 and Arg1016
play ancillary roles. Multiple conformations observed for
His1023 and adjacent residues suggest that binding of A3 to
collagen involves an induced-fit mechanism.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the staff of the EMBL Outstation DESY (Hamburg, Germany) for assistance in x-ray data collection.
![]() |
FOOTNOTES |
---|
* This work was financially supported by the Council of Medical Science program (Grant 902.26.193), by the Council for Chemical Science program (Grant 326-026) from the Netherlands Organization for Scientific Research, and by European Union support of the work at EMBL (Hamburg, Germany) through the HCMP Access to Large Installations Project (Contract CHGE-CT-0040).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF286587.
The atomic coordinates and the structure factors (code 1FE8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Both authors contributed equally to this work.
To whom correspondence should be addressed: Tel.:
31-30-250-7230; Fax: 31-30-251-1893; E-mail:
E.Huizinga@chem.uu.nl.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M006548200
2 The atomic coordinates for the crystal structures of free A3 were taken from PDB entries 1AO3 and 1ATZ.
3 E. G. Huizinga, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: vWF, von Willebrand factor; CK, cystine knot; PBS, phosphate-buffered saline; BSA, bovine serum albumin; a.u., asymmetric unit; CDR, complementary determining region; ELISA, enzyme linked immunosorbent assay; fur-BHK, baby hamster kidney cells overexpressing furin; MIDAS, metal ion dependent adhesion site; n.c.s., noncrystallographic symmetry; Se-met, selenomethionine; wt, wild-type.
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