von Willebrand Factor (vWF) is a multimeric
protein that mediates platelet adhesion to exposed subendothelium at
sites of vascular injury under conditions of high flow/shear. The A1
domain of vWF (vWF-A1) forms the principal binding site for platelet glycoprotein Ib (GpIb), an interaction that is tightly regulated. We
report here the crystal structure of the vWF-A1 domain at 2.3-Å resolution. As expected, the overall fold is similar to that of the
vWF-A3 and integrin I domains. However, the structure also contains N-
and C-terminal arms that wrap across the lower surface of the domain.
Unlike the integrin I domains, vWF-A1 does not contain a metal
ion-dependent adhesion site motif. Analysis of the
available mutagenesis data suggests that the activator botrocetin binds
to the right-hand face of the domain containing helices
5 and
6.
Possible binding sites for GpIb are the front and upper surfaces of the
domain. Natural mutations that lead to constitutive GpIb binding (von
Willebrand type IIb disease) cluster in a different site, at the
interface between the lower surface and the terminal arms, suggesting
that they disrupt a regulatory region rather than forming part of the
primary GpIb binding site. A possible pathway for propagating
structural changes from the regulatory region to the ligand-binding
surface is discussed.
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INTRODUCTION |
von Willebrand Factor
(vWF)1 is a multimeric
protein that mediates platelet adhesion to exposed subendothelium at
sites of vascular injury (1). The adhesive properties of vWF are
tightly regulated so that plasma vWF does not normally interact with
circulating platelets. vWF, however, will bind to platelets after it is
"activated" by poorly understood conformational changes that occur
after it binds to the vessel wall. A reduction in the plasma
concentration of vWF or mutations that impair binding, activation, or
assembly of vWF multimers cause von Willebrand disease (vWD), a common bleeding disorder characterized by decreased platelet adhesion and
mucocutaneous bleeding (2).
vWF-mediated adhesion of platelets to the vessel wall, under the high
flow/shear conditions present in circulating blood, is mediated by
sequences within the first (A1 domain) and third (A3 domain) A type
repeats of vWF. The A1 domain (residues 479-717) binds to platelet
glycoprotein Ib·IX·V complex (GpIb), subendothelial heparans, cell
surface sulfatides (reviewed in Ref. 3), and the non-fibrillar collagen
type VI (4). The vWF-A3 domain contains the principal site for binding
the fibrillar collagens types I and III (5, 6).
Although initially noted in the primary sequence of vWF, the A domain
has been subsequently discovered in a large number of cell
matrix-associated or adhesive proteins and receptors (7). For example,
varying numbers of A domains are found in several of the atypical,
short chain collagens. A single A domain is inserted into the sequence
of several integrin receptors, where it is generally referred to as the
I domain. A/I domains are frequently involved in either cell adhesion
or cell ligand interactions. In 1995, we reported the crystal structure
of the first family member, the I domain of the leukocyte integrin
M
2 (8), and the crystal structures of several A domains have now
been solved (9-12). This work has provided new insights into how A
domains mediate cellular adhesion and facilitates detailed
structure-function studies. All A domains have a very similar structure
comprising a variant of the dinucleotide-binding fold. The integrin I
domains contain a metal ion-dependent adhesion site (MIDAS)
on the upper face of the domain that is an important element of ligand
binding (8, 13-15). In contrast, the vWF-A3 domain does not bind metal
and does not require metal for binding to collagen (10).
To advance studies of vWF binding to platelet GpIb and to gain more
understanding of the molecular switches that activate vWF, we have
solved the crystal structure of the vWF-A1 domain and in this paper
correlate its structure with existing biochemical and mutational
data.
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EXPERIMENTAL PROCEDURES |
Purification and Crystallization--
Recombinant vWF-A1
containing residues 475-709 of mature vWF and 12 residues at the N
terminus from the expression vector (MRGSHHHHHHGS) was expressed in
Escherichia coli, refolded, and purified as follows. Our
previously published technique (16) was used to transform cells, induce
protein, and harvest inclusion bodies. Next, the washed pellet was
solubilized by the addition of 6.5 M guanidine
hydrochloride in 50 mM Tris-HCl, pH 7.5. The solubilized
protein was diluted 40-fold in 50 mM Tris-HCl, 500 mM NaCl, 0.2% Tween 20, pH 7.8. It was passed over an
Ni2+-chelated Sepharose (Pharmacia) column equilibrated
with 25 mM Tris-HCl, 200 mM NaCl (pH 7.8)
buffer. vWF-A1 protein eluted from the column with 350 mM
imidazole. The isolated protein was absorbed to and eluted from a
Heparin-Sepharose column (Amersham Pharmacia Biotech). The highly
purified protein was dialyzed against 25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.8. This protein failed to
produce crystals suitable for x-ray analysis.
The protein (0.4 mg/ml) was next treated with immobilized
-chymotrypsin (Sigma) in 0.1 M Tris, pH 8.0, 0.15 M NaCl, 0.1% Triton X-100 for 24 h at 4 °C with
constant agitation, loaded onto a Heparin-Sepharose column (Pharmacia)
equilibrated with 0.1 M Tris, pH 8.0, 0.15 M
NaCl, and then eluted with 0.1 M Tris, pH 8.0, 0.6 M NaCl. Finally, the protein was diluted 3-fold with water
and concentrated to 4 mg/ml. The molecular mass estimated by
SDS-polyacrylamide gel electrophoresis reduced from 27 to 24 kDa after
chymotrypsin digestion. In the crystal structure (see below), the C
terminus is ordered to within 4 residues of the C terminus of the
expressed domain, and at the N terminus the first residue visible in
the electron density map is Asp498. Chymotrypsin cleaves
specifically after aromatic residues, and Tyr495 at the N
terminus is the only aromatic residue that is not visible in the final
electron density map. Cleavage after Tyr495 and the loss of
33 residues from the N terminus gives a predicted size of 24.5 kDa,
consistent with the size estimated by SDS-polyacrylamide gel
electrophoresis. Other aromatic residues are presumably protected from
cleavage by the folded conformation of the domain. Cleavage did not
detectably perturb vWF-A1 binding to GpIb or its ability to inhibit
ristocetin-induced platelet aggregation by full-length vWF (data not
shown).
Crystals of the chymotrypsin-cleaved protein were grown by hanging drop
vapor diffusion using 0.1 M Tris, pH 8.5, 8% polyethylene glycol 8000. Larger crystals grew to dimensions 0.1 mm × 0.1 mm × 1.0 mm in the presence of 5 mM CdCl2
and belong to the space group P61 with cell dimensions
a = b = 86.4 Å, c = 68.1 Å,
=
= 90°,
= 120°. The asymmetric unit contains one vWF-A1 domain and 57%
solvent.
Data Collection, Structure Determination, and Refinement--
A
single crystal was transferred into a cryoprotectant buffer consisting
of 0.1 M Tris, pH 8.5, 5% polyethylene glycol 8000, 30%
glycerol, and frozen by immersion in a stream of nitrogen at 100 K. Data were collected with a Rigaku RU-200 x-ray generator, focusing
mirrors, and an R-AXIS IV imaging plate. Data were reduced with DENZO
(17) and scaled with SCALEPACK (17) with an Rmerge of 8.3%
and 91% completeness to 2.3 Å resolution. Subsequent calculations were performed using the CCP4 suite of programs (18) unless otherwise
noted. A mercury derivative was prepared by soaking crystals in the
cryoprotectant buffer plus 5 mM HgCl2 for
48 h. A 2.8-Å data set was collected from this derivative with an
Rmerge of 13.4%, a mean isomorphous difference with the
native data set of 26.7%, and 99.8% completeness.
Molecular replacement was performed using the crystal structure of the
integrin
2-I domain (11), stripped of loops and side chains, as the
search model. The highest peak in the cross-rotation function gave the
correct orientation of the monomer. The translation function gave a
stronger top peak in space group P61 than in
P65, establishing the correct enantiomorph. Rigid body
refinement using XPLOR (19) led to an R factor of 52% and a
correlation coefficient of 0.45. Using the mercury derivative data and
model-derived phases, a difference Fourier revealed the position of two
mercury atoms. These sites were refined using HEAVY (20), and phases
were calculated using MLPHARE (phasing power = 0.41), leading to
an experimental map that was solvent flattened using DM. The heavy
atom-derived and molecular replacement-derived maps were then averaged
using the program RAVE (21). This averaged map was of higher quality than either of the component maps and was readily interpretable. An
initial round of model building using program O (22) allowed insertion
of 173 residues including some side chains. This model was subjected to
positional refinement using XPLOR, resulting in an R factor
(RWORK) of 42.3% for the working set and an
RFREE (calculated on 10% of reflections omitted from
refinement) of 47.2% at 2.8 Å resolution. Further rounds of
positional and B-factor refinement, model building, and extension of
the data to 2.3 Å resolution led to an RWORK of 24.3% and
RFREE of 31.2%. An anomalous difference Fourier calculated
at this stage revealed the presence of an anomalous scatterer (peak
height, 4.6
), presumed to be Cd2+, coordinating
histidines His559 and His563 in the
B-
C
loop. Water molecules were next added at the positions of Fo
Fc peaks greater than 3
, where reasonable hydrogen-bonding
partners existed. After applying a bulk solvent correction, the final
R-factor was 19.2% for all data between 10 and 2.3 Å (RFREE = 23.8%); the model includes residues 498-705, 93 water molecules, and one Cd2+ ion. All main chain torsion
angles lie within the most favored (88.8% of residues) or additional
allowed regions (11.3% of residues) of the Ramachandran plot as
defined in PROCHECK. There are no cis-prolines. The RMS deviations from
ideal bond lengths and angles are 0.011 Å and 1.43°, respectively.
 |
RESULTS |
Structure of the vWF-A1 Domain--
We crystallized a recombinant
vWF-A1 domain and solved its structure at 2.3 Å resolution (Table
I). The overall fold is, as expected,
very similar to the von Willebrand Factor A3 domain and the
integrin I-domain, with a central hydrophobic parallel
-sheet
flanked on two sides by amphipathic helices (Figs. 2 and 3). Helices
are labeled 1-7 and strands A-F by homology with the
M
2 I
domain (8). There is no equivalent of the second helix,
2, found in
the
M and
L I domains. The disulfide bridge linking the N- and
C-proximal sequences (Cys509-Cys695) is well
ordered. In addition, ordered structure extends 11 residues N-terminal
to, and 10 residues C-terminal to, the disulfide bridge.
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Table I
Summary of crystallographic analysis
Space group P61, a = 86.4 Å, b = 86.4 Å, c = 68.1 Å, = = 90.0°, = 120.0° 1 molecule in the
asymmetric unit; solvent content 57%. Refinement statistics:
resolution, 10-2.3 Å; RWORK = 0.196; RFREE = 0.238;
No. of waters = 93; RMSD bond lengths = 0.011 Å; RMSD
angle = 1.43°.
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The domain is 40 Å high, 30 Å wide across the
-sheet, and 40 Å broad. The overall shape is cuboid, with six fairly flat faces; four of
these are shown in Fig. 4. (a) The "front" face lies at one edge of the
-sheet and includes side chains from helices
3
and
4, strand
C, and their connecting loops. This face also includes part of the salt-bridge network (see below). (b)
The "upper" face lies at the C-terminal end of the
-sheet and is composed of loops connecting the sheet to the flanking helices. This
face contains two positively charged residues, Arg524 and
His656, packed together at the center of an otherwise polar
but uncharged surface ~20 Å in diameter, with charged groups at the
periphery. In integrin I domains, this upper face contains the MIDAS
motif, consisting of three closely apposed loops that together
coordinate a magnesium ion. However, the homologous loops of vWF-A1 are
not suitable for metal coordination. (c) The "lower"
face includes the N- and C-terminal arms and the loops connecting the
-helices to the N-terminal end of the
-sheet. (d) The
"right-hand" face includes helices
4,
5, and
6 flanking
one side of the
-sheet, and is highly basic. (e) The
"left-hand" face (not shown) includes helices
1,
3, and
7
flanking the opposite side of the
-sheet and is highly acidic.
(f) The "back" face (not shown) includes helices
6,
7, and strand
F.
Salt Bridges and Buried Charges--
The vWF-A1 domain contains a
large number of charged amino acids. Some of these form an elaborate
salt-bridge network that wraps around the lower rim of the domain (Fig.
4a). These stabilizing salt bridges may explain the
sensitivity of this region to alanine mutagenesis (23). There are four
buried acidic residues: (a) Glu557 from strand
B forms stabilizing salt bridges with two histidine side chains
(His559 and His563) from the
B-
C hairpin;
(b) Glu626 is buried but sits at the N-terminal
end of helix
4, where it may be stabilized by the helix dipole;
(c) Asp514 at the bottom of strand
A is
buried and is stabilized by salt bridges to Arg552 and
Arg611, which form part of the salt-bridge network. A
buried salt bridge between an aspartic acid from the bottom of strand
A and an arginine from the
4-
D turn is seen in all of the A/I
domain structures that have been studied, suggesting that this is an
important element of folding; (d) Asp520 is
buried below the upper face of the domain without a stabilizing salt-bridge partner. This residue is homologous in sequence to the
aspartic acid of the DxSxS integrin MIDAS motif; it has been suggested
that the side chain of Arg524 might form a salt bridge with
the buried aspartic acid, substituting for the metal ion, but our
crystal structure shows that instead Arg524 points outward
into solution, packing against the side chain of His656
from the
E-
6 loop.
N- and C-terminal Arms and von Willebrand Disease Type IIb
Mutations--
Upstream from the N-terminal strand,
A, the chain
makes a 90° turn, with Cys509 three residues from the
turning point. Further upstream, the side chains of Phe507,
Tyr508, and Ile499 pack against hydrophobic
elements on the surface of the domain, and His505 and
Glu501 make stabilizing salt bridges with helices
1 and
3. At the C terminus, the
7 helix extends a turn beyond the
disulfide bridge, followed by an extended structure that packs against
the hydrophobic side of the domain as far as Ala701; in
addition, Glu700 makes a salt bridge with
Arg511 near the N terminus. Beyond this, a sequence of
three prolines in an extended conformation protrudes downward from the
body of the domain.
Ordered electron density exists for all residues that have been
identified as natural mutation sites leading to the type IIb phenotype
("gain of function," constitutive binding to GpIb). All of these
sites lie on the lower surface of the domain at the interface between
the body of the domain and the N- and C-terminal arms (see Table
II and Fig. 3). Our crystal structure
shows that in the wild-type protein, these residues are all involved in
salt bridges or hydrophobic packing. The most likely effect of these mutations is that they disrupt the interface between the N- and C-terminal arms and the body of the domain. Scanning mutagenesis has
led to the identification of further mutants with a similar phenotype
(23). These lie in the same region and include a triple-alanine mutation in the middle of the C-terminal helix
(RDE687-689), which breaks salt-bridge contacts with helix
1 and the salt-bridge network. Mutations of Cys509 to
Gly or Arg, which break the disulfide bridge, also lead to constitutive
GpIb binding (24).
Comparison with the vWF-A3 Domain--
The central
-sheets of
the A1 and A3 domains overlap very closely, with an RMS deviation on
main chain atoms of 0.55 Å for 40 residues (similar comparisons with
the integrin I domains also give values in the range 0.5-0.6 Å).
Extending the overlap to the entire domain gives an RMS deviation of
1.4 Å for 165 residues in equivalent structural environments, as
defined in MULTIFIT (25). The resulting alignment has 21% amino acid
identity and is shown in Figs. 1 and
2. The A3 crystal structure lacks the N-
and C-terminal extensions found in A1, but the disulfide bridge is
similarly located. The
helices are generally similar in length and
orientation, with the major differences restricted to the loops
connecting strands and helices on the upper and lower surfaces of the
domain. vWF-A1 and vWF-A3 both lack the
2 helix found in the
integrin
M
2 and
L
2 I domains. In A1, helix
7 is preceded by a turn of 310 helix, whereas in A3, the 310
helix is replaced by an
helix that is longer by 3 residues. The
surface charge distribution is less asymmetric in A3 than in A1,
lacking the basic patch on the right-hand face of A1.

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Fig. 1.
Alignment of the sequences of the vWF A1 and
A3 domains based on their three-dimensional structures.
Non-homologous regions are shown in lowercase letters.
Secondary structure assignments are shown for the A1 domain. Helices
are labeled 1-7 and strands A-F by homology with the M I domain
(8). There is no equivalent of the second helix, 2, found in the
M and L I domains. Open boxes indicate stretches of
310 helix.
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Fig. 2.
Stereo C plot comparing vWF-A1
(solid lines) with vWF-A3 (dashed lines).
The two molecules have been superimposed using MULTIFIT (25). The N and
C termini of vWF-A1 are labeled. Every 10th residue (starting at 506)
is shown as a small circle, with occasional numbering. The
N- and C-proximal cysteines forming the disulfide bridge are shown as
large circles.
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The shape of the upper surface of the domain is affected by three
changes in the surface loops. (a) At the top of the
A
strand, arginine Arg524 in A1 points out into solution,
whereas the equivalent A3 residue, Ser938, points inward.
The side chain of the next residue, Leu525, points into the
interior of the protein; homologous residues in the integrin I domains
are similarly oriented. This contrasts with A3, in which
Phe939 points out into solution. These two differences give
the
A-
1 turn a quite different conformation in A1 than in A3.
(b) There is a 1-residue insertion in the
3-
4 loop of
A1, which wraps over the top of the
B-
C hairpin, allowing space
for the side chain of His559 that replaces a glycine in A3.
(c) A1 has a 4-residue insertion in the
D-
5 loop,
which includes a turn of 310 helix. The lower surface is
affected by the following changes: (i) in the
C-
3 loop,
Leu568 packs more closely into the hydrophobic core than
the larger tryptophan in A3, causing a shift of the entire loop; (ii)
the end of helix
4 has a different conformation, and the
4-
D
loop is two residues shorter in A1; (iii) The
6-
F turn adopts a
different conformation.
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DISCUSSION |
Using our crystal structure of the vWF-A1 domain, coupled with the
analysis of naturally occurring and experimentally introduced mutations, we can begin to map the binding sites for GpIb and activators like botrocetin and ristocetin. In addition, with an understanding of the type IIb vWD "gain-of-function" mutations in
relation to the ligand-binding surfaces, we can begin to formulate models of the activation process.
Botrocetin and Ristocetin Binding Sites--
Two groups have
performed scanning mutagenesis on the A1 domain (23, 26) which can be
used to help localize the binding sites for these activators.
Matsushita and Sadler (23) measured the direct binding of botrocetin to
vWF. Ignoring those mutations that involve buried charges and probably
cause misfolding of the A1 domain, the remaining mutation sites leading
to reduced (<50% normal) binding are located in helices
5 and
6
and adjoining structures: Arg636 in helix
5,
Arg629 and Arg632 in the 310 helix
immediately preceding
5, and RLIEK663-667 in the
neighboring
6 helix. These data strongly suggest that botrocetin
binds to the right-hand face of the domain (Figs.
3 and 4).
Mutation of the lysine cluster KKKK642-645 also reduced
botrocetin binding (23), but Kroner and Frey (26) report normal
botrocetin-induced function for this mutant.

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Fig. 3.
Main chain schematic of the vWF-A1 domain,
with -strands (arrows) and helices (coils)
(drawn with MOLSCRIPT, RASTER3D, and RENDER (32-34)). The two
cysteines involved the disulfide bridge are shown as yellow
spheres. Sites of von Willebrand disease type IIb mutations (both
natural and induced) are shown as red spheres. Mutants with
reduced botrocetin binding are in green. Mutations with
selective loss-of-function (reduced ristocetin-induced binding but
normal botrocetin-induced binding) are in cyan (23) or
black (26), and a mutant with reduced GpIb binding but
normal botrocetin binding is in blue (23). The mutation of
KKKK642-645 in the 5- E loop also reduces binding to
heparin (26). For multiple site mutants, spheres are placed
near the midpoint of the mutation.
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Fig. 4.
Four faces of the vWF-A1 domain, defined from
the orientation in Fig. 3. Atoms are shown as spheres,
drawn with BALLS, RASTER3D, and RENDER (33, 34). Arginines and lysines
are in blue, aspartic acids and glutamic acids are in
red, and histidines are in green; all other
residues are in gray. The proposed binding site for
botrocetin includes the residues marked "B," and a possible binding
site for heparin is circled. a, front face (same
view as Fig. 3). Sites of mutations with impaired GpIb binding
(Gly561, Glu596, and Lys599) are
labeled. b, upper face at the C-terminal end of the
-sheet. c, lower face including the N-terminal arm.
d, right-hand face, including helices 5 and 6.
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Matsushita and Sadler (23) reported four mutants with a selective
loss-of-function: loss of ristocetin-induced GpIb binding but normal
botrocetin-induced binding. These map near to the upper face of the
domain (Fig. 3) and consist mainly of buried (Glu626,
Asp520) or partly buried charges (Lys534 salt
bridges to Glu531). These mutations, which lead to a loss
of stabilizing salt bridges, may cause local instability in the upper
surface of the domain that disrupts GpIb binding. The natural mutant
(Gly561
Ser) has the same phenotype (27).
Gly561 is part of the tight
B-
C turn at the upper
front/edge of the domain. Its main chain torsion angles are not
unusual, and modeling suggests that the mutant side chain can point out
into solution without severe distortion of the main chain. The ability
of botrocetin to overcome the binding deficiency in these mutants may
arise from the stabilizing effect of its tight binding to an adjacent surface of the A1 domain. Kroner and Frey (26) reported three further
mutations with the same phenotype, in the region of the type IIb
mutations and the lysine cluster KKKK642-645, adjacent to
the N- and C-terminal arms. The arms contain proline-rich segments that
have been implicated in ristocetin binding (28). It is therefore
possible that selective loss-of-function in these mutants arises from
defective ristocetin binding.
The GpIb Binding Site--
Data from various sources, when linked
together, provide some clues to the location and extent of the GpIb
interaction site within the A1 domain. First, previously reported
studies from our laboratory on the functional properties of a vWF-A1/A3
chimera that contains the N-terminal half of vWF-A1 (as far as
Leu598 in the middle of helix
4), but still binds GpIb
normally (6), strongly suggest a role for the front half of the domain.
Second, the majority of mutations that lead to a selective
loss-of-function (described above) cluster at or near the upper/front
surface of the domain, in contrast to the gain-of-function mutants,
which cluster on the lower surface (see below). Third, Matsushita and Sadler reported that the double alanine mutation at Glu596
and Lys599 on the front surface (helix
4) showed reduced
GpIb binding (without affecting botrocetin binding) and suggested that
it might form part of the GpIb binding site. Two further natural
mutations (Type IIm) with impaired GpIb binding lie in helix
4 and
the following loop, but both residues (Phe606 and
Arg611) are buried in our crystal structure and the
mutations probably lead to destabilization of the folded structure.
Overall, the data point to a role for the upper/front surfaces of the
domain in GpIb binding, but further work is clearly required to confirm this location.
Type IIb Mutations and Regulation of GpIb Binding--
The vWD
type IIb mutations, which lead to constitutive binding of vWF to GpIb,
are all located at or near the interface between the lower surface of
the domain and the N- and C-terminal arms (Fig. 3), and the mutations
are expected to break salt bridges and hydrophobic contacts that
stabilize this interface. Because these are so numerous, are
"gain-of-function," and map to both sides of the interface, it is
very unlikely that this region forms a binding surface for GpIb;
rather, it is more likely to be involved in the regulation of binding
affinity. The separation between this region and the most likely GpIb
binding surface begs the question of how structural changes are
communicated between the two sites.
A possible clue comes from studies on the homologous integrin I domain.
Whereas ligand binding sites have been shown to lie on the upper
surface of the domain, Zhang and Plow (29) have made
M/
L I
domains chimeras, in which the swapping of sequences in the lower
surface of the domain (at sites homologous to the type IIb mutations in
vWF-A1) leads to constitutive high affinity ligand binding. A plausible
pathway for communicating structural changes from one face of the I
domain to the opposite face is provided by the two crystal structures
of the integrin
M I domain observed by Lee et al. (30).
In these structures, a change in the shape and charge distribution of
the upper ligand-binding face that would influence its affinity for
ligand is propagated via a large (10 Å) downward shift of the
C-terminal helix,
7, to the lower face of the domain. The more
extended conformation of the domain was identified with the high
affinity state.
Although we have no evidence for such a conformational change in the
vWF-A1 domain, we note that several type IIb mutations map either to
the C-terminal helix or to a residue that salt-bridges to the helix
(Table II). We expect that these mutations disrupt salt bridges and
hydrophobic interactions that lock the helix against the body of the
domain. Although the N and C termini remain linked by a disulfide
bridge in type IIb vWD, a substantial downward shift of the C-terminal
helix is theoretically possible, requiring a concerted movement of the
N-terminal arm. Thus, by destabilizing the folded conformation of the
terminal arms, the type IIb mutations could shift the equilibrium
toward the extended conformation. A conformational switch of this kind,
in which the high affinity state is identified with a more extended
conformation of the A1 domain, could be triggered when high flow/shear
unfolds immobilized vWF (31).
We thank Laurie Bankston for fruitful
discussions and Remy Loris for assistance in refinement.