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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2032-2044
Molecular Modeling of Ligand and Mutation Sites of the Type A Domains
of Human von Willebrand Factor and Their Relevance to von Willebrand's
Disease
By
P. Vincent Jenkins,
K. John Pasi, and
Stephen J. Perkins
From the Katherine Dormandy Haemophilia and Haemostasis Centre,
Department of Haematology and the Department of Biochemistry and
Molecular Biology, Royal Free Hospital and School of Medicine, London,
UK.
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ABSTRACT |
von Willebrand factor (vWF) is a large multimeric, multidomain
glycoprotein found in platelets, endothelial cells and plasma. The A1,
A2, and A3 domains in vWF mediate binding to glycoprotein Ib,
ristocetin, botrocetin, collagen, sulphatides, and heparin and
provide a protease cleavage site. Mutations causing types 2B, 2M, and
2A von Willebrand's disease (vWD) are located in the A1 and A2
domains. Homology modeling was performed to provide a molecular
interpretation of vWF function and mutation sites. This was based on
our previous alignment of 75 vWF-A sequences, the doubly wound  /
fold seen in recent vWF-A crystal structures from complement receptor
type 3 and lymphocyte function-associated antigen-1, and our new
alignment of 28 vWF A1 and A2 sequences from different species. The
active site in doubly-wound / folds forms a crevice that is
located at the switch point between the two halves of the central
-sheet, and usually contains two metal-binding Asp residues in the
vWF-A superfamily. Although one of these Asp residues is absent from
the A1, A2, and A3 domains, this crevice is shown to correspond to the
ristocetin binding site in the A1 domain and the protease cleavage site
in the A2 domain. The residues R571-K572-R578-R579-K585 are found to be
conserved in 28 A1 sequences and are predicted to constitute the
heparin binding site in the A1 domain. Inspection of the type 2M vWD
mutation sites that are involved in downregulation of glycoprotein Ib
(GpIb) binding to vWF shows that these are spatially clustered at the
carboxyl-edge of the -sheet and above it in the A1 domain and may
directly perturb GpIb binding. In contrast, the type 2B vWD mutation
sites that are involved in upregulation of GpIb binding to vWF are
spatially clustered at the amino edge of this -sheet and below it
and are located on the opposite side of the A1 domain from the type 2M mutation sites. The type 2B mutations are located between the heparin
and GpIb binding sites. Because heparin binding inhibits the
interaction with GpIb, this provides an explanation of vWF upregulation. The type 2A vWD mutation sites in the A2 domain correspond to buried residues that are otherwise 100% conserved across
all 28 species, and are likely to be important for the correct folding
of the A2 domain and its physiologically important protease site.
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INTRODUCTION |
VON WILLEBRAND FACTOR (vWF) is a large
glycoprotein that is found in plasma and platelets and is synthesized
by megakaryocytes and endothelial cells.1-3 vWF plays at
least two essential roles in hemostasis. It is involved in platelet
adhesion to the damaged vascular endothelium, and it stabilizes factor
VIII in plasma by acting as its carrier molecule. Precursor vWF
contains 13 domains in its monomeric structure, which are multiples of
four domain types A to D in the arrangement
D1-D2-D -D3-A1-A2-A3-D4-B1-B2-B3-C1-C2.4 After
synthesis, the precursor is modified by post-translational processing,
in which it dimerizes, and cleavage of the D1-D2 domain pair occurs.
The monomer contains 2,050 residues of molecular weight 220,000. Multimeric vWF contains between 2 to 100 subunits with molecular
weights ranging up to 20 ×106.
The A1, A2, and A3 domains mediate key macromolecular interactions by
vWF. The A1 domain corresponds to residues 497 to 716 in mature vWF,
whose N- and C-termini are joined by a disulfide bridge at Cys509 and
Cys695. It is involved in vWF binding to the platelet receptor
glycoprotein Ib (GpIb). This is the primary mechanism by which vWF
binds to platelets under conditions of high shear stress. vWF does not
bind spontaneously to GpIb. Conformational changes are thought to
expose the vWF-A1 domain after the binding of other regions of vWF to
collagen on the subendothelium. In vitro tests show that vWF can be
stimulated to bind to platelets by the antibiotic ristocetin (molecular
weight of several thousands) and two forms of the snake venom
derivative botrocetin (molecular weight 25,000 and 26,500), although
these two compounds involve different residues in the vWF-A1
domain.5 The vWF-A1 domain also contains distinct binding
sites for heparin and sulphatides and a further site for
collagen.2,4 The binding of vWF to collagen permits its
interaction with GpIb, whereas vWF binding to heparin inhibits it. The
vWF-A2 domain spans residues 717 to 909 and does not contain a
disulfide bridge between its N- and C-termini (although there is a
bridge between Cys906 and Cys907). It contains a physiological cleavage
site between Tyr842 and Met843 that is associated with a
metalloprotease in normal circulation.6,7 The vWF-A3 domain
spans residues 910 to 1111 in which its N- and C-termini are joined by
a disulfide bridge between Cys923 and Cys1109.8 This
contains the major binding site for fibrillar collagens.9,10
von Willebrand's Disease (vWD) is the most common inherited bleeding
disorder, in which type 2 vWD corresponds to functional abnormalities
in vWF (as opposed to a deficiency or absence of vWF in types 1 and 3 vWD, respectively).11-13 A large number of mutated residues
that give rise to three variant types 2A, 2B, and 2M vWD have been
found in the vWF-A1 and vWF-A2 domains. Type 2B vWD is characterized by
the loss of high molecular weight multimers of vWF as a result of an
increased affinity of vWF for GpIb, and shown in vitro by increased
ristocetin-induced binding of vWF to platelets. Most type 2B vWD
mutations involve a short segment in the vWF-A1
domain.11,14,15 Type 2M vWD is characterized by a normal
pattern of vWF multimers but a decreased binding of vWF to GpIb and a
decreased ristocetin-induced binding of vWF to platelets. Mutations
causing type 2M vWD are also located in the vWF-A1
domain.16-19 Type 2A vWD results from the loss of high and
intermediate vWF multimers, either through the impaired intracellular transport of vWF multimers (group I mutations) or the abnormal proteolytic degradation of vWF (group II mutations).20 Type 2A mutations arise mainly within the vWF-A2 domain.11,21-23
Three-dimensional structures are required to provide a molecular
interpretation of vWF function and a molecular explanation of the
mutations involved in type 2 vWD. The vWF-A domains belong to a large
superfamily that include other plasma proteins, collagens, integrins,
and other extracellular proteins that mediate cell-cell, cell-matrix,
and matrix-matrix interactions.24,25 The vWF-A protein fold
was first identified by our structure prediction analysis, and this
included an initial consideration of vWF mutation sites.26,27 Crystal structures of the vWF-A domains in
human complement receptor type 3 (CR3) and the leukocyte integrin
lymphocyte function-associated antigen-1 (LFA-1) have since been
determined to confirm the prediction.28-31 Since then, vWF
sequences from 28 different mammalian species from 15 placental orders
are now available.25,32 The combination of our vWF-A
predictions with these crystal structures and new sequences now make it
possible to create homology models for the human vWF-A1, -A2, and -A3
domains and assess them. We show that major functional sites in the A1 and A2 domains correspond to the classic active site region of this
protein fold. Recent crystal structures of protein-heparin complexes33 permit a set of conserved residues in vWF-A1 to be identified as a heparin binding site. The type 2B vWD mutations are
shown to be located between the heparin and GpIb binding sites and this
provides an explanation of how vWF binding is upregulated, whereas
those involved in type 2M vWD are shown to be directly associated with
the GpIb binding site and explain how vWF binding is downregulated. The
vWF-A2 mutations that lead to type 2A vWD are shown to correspond to
buried conserved residues, and these are discussed also.
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MATERIALS AND METHODS |
Crystal structures in the vWF-A superfamily.
Molecular graphics models of the three vWF-A1, -A2, and -A3 domains in
human vWF were constructed as follows. The multiple sequence alignment
of 75 vWF-A sequences25 was used to align the three vWF-A
sequences to be modeled with the crystal structures of CR3 (CD11b/CD18)
and LFA-1 (CD11a/CD18), and this is summarized in
Fig 1 using the same sequence numbering as
in Perkins et al.25 Residues L512-Y693 of the vWF-A1 domain
(out of residues 479 to 716), V733-Q904 of the vWF-A2 domain (out of
residues 717 to 909), and P926-F1104 of the vWF-A3 domain
(out of residues 910 to 1111) could be modeled. All three models were
based on the crystal structure of the Mg-bound CR3 vWF-A
domain28 (Brookhaven code 1ido). As a control of this
structure, the 1ido coordinates were compared with those for Mn-bound
CR329 (code 1jlm) and for LFA-1,30,31 which
correspond to metal-free LFA-1 (code 1zon), Mg-bound LFA-1 (code 1zoo),
and Mn-bound LFA-1 (code 1zop) in Fig 1. A conformational change has
been reported between Mg-bound and Mn-bound CR3 that involves the metal
site, the -helix A7, and three of the five loops on the
carboxyl-edge of the -sheet.29 Only -helix A7 moves
in metal-free LFA-1, which has a structure otherwise identical to
Mg-bound and Mn-bound LFA-1, the latter pair having identical
structures.31 The DSSP program34
was used to assign secondary structure in the crystal structures.
Side-chain solvent accessibilities were calculated by the method of Lee
and Richards using a probe diameter of 1.4 Å in the COMPARER
program.35,36 The secondary structures and accessibilities of 1zon, 1zoo, and 1zop were indistinguishable, and
their mean is shown in Fig 1. Figure 1 shows that the secondary structures of LFA-1 and both forms of CR3 are very similar with the
exception of changes in the 310-helix (G) and -helix (H) content of A7 and minor ones in A6. Likewise, if the sidechain accessibilities were classified as exposed (values of 2 to 9) or
inaccessible (0 to 1), Fig 1 shows that the three crystal structures are very similar except for small changes at two carboxyl-edge loops
connecting BD with A5 and BE with A6, and the C-terminal -strand BF
and -helix A7. These differences are negligible in terms of the
analyses of the ristocetin binding site and the types 2B, 2M, and 2A
vWD mutations below.
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| Fig 1.
Sequence alignment for human CR3 and LFA-1 and
the vWF-A1, -A2, and -A3 domains. The vWF sequence numbering is
indicated at the start and end of each vWF-A sequence. The -helix
and -strand secondary structures in the CR3 and LFA-1 crystal
structures and the three vWF-A models are denoted by A1 to A7 and BA to
BF, respectively. Residues that were rebuilt as new loops are denoted
in bold and underlined. The DSSP analysis is represented as follows: E,
-strand; B, single residue -ladders; T, turn; G, 310
helix; and H, -helix. The COMPARER sidechain solvent accessibilities
are on a scale of 0 to 9 for each residue, where 0 corresponds to 0%
to 9% solvent exposure, 1 to 11% to 19% solvent exposure, and so on,
and buried residues have accessibilities of 0 or 1.
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Construction of vWF-A homology models.
Protein structures were visualized and modeled using INSIGHT II 95.0 software with HOMOLOGY and DISCOVER modules (Biosym/MSI, San
Diego, CA) on INDY Workstations (Silicon Graphics, Reading, UK). The rigid body fragment assembly method used in
HOMOLOGY was used to construct the three models. A conserved core of
138 to 156 -helix, -strand and loop residues was defined based on the 1ido crystal structure. In the vWF-A1 model, the remaining 26 residues at five surface loop regions were modeled from a database of
protein fragments using the Brookhaven loop database of INSIGHT II.
These were between L544-K549, G588-V591, S607-P612, V635-Q639, and
P655-L659 where sequence insertions or deletions occurred (Fig 1). In
the vWF-A2 model, 34 residues in six loops were modeled at M765-D770,
Y807-T813, H825-V829, D851-G858, N870-V873, and W881-A884. In the
vWF-A3 model, 29 residues in five loops were modeled at K957-L964,
G1003-G1008, M1022-G1024, A1051-T1056, and D1066-A1071. All the
sidechains were replaced according to the human vWF-A sequences. Energy
refinements using DISCOVER were performed at the loop splice junctions,
then on the loop regions, and finally the mutated core residues. The
core residues were tethered to their original positions with greater
energy terms than the reconstructed loops. The refinements were based
on the consistent valence forcefield, and iterations were made using a
combination of the steepest descent and conjugate algorithms. These
improved the connectivity of the model and reduced the proportion of
bad contacts or stereochemistry as confirmed by the use of the PROCHECK
program.37 The vWF mutation residues in the
vWF-A1 and vWF-A2 domains are summarized in
Table 1. The site of each one was
identified by a sphere at the -carbon atom in the vWF-A models of
Fig 6.
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| Fig 6.
Location of the types 2B, 2M, and 2A mutation sites in
homology models of the vWF-A1 and vWF-A2 domains. Each of the vWF-A1 and vWF-A2 domains is shown in two orthogonal perspectives rotated by
90° along the vertical axis. The central -sheet is seen edge-on in both views. The mutation residues are identified by spheres at their
-carbon atom. The protein backbone is represented by a ribbon. The
eleven residues deleted in the type 2M "Milwaukee" mutation are
shown as the cyan ribbon in the vWF-A1 model.
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Electrostatic determination of the surfaces was performed using DELPHI
(Biosym/MSI).38 The total electrostatic energy of the
system was calculated using a full coulombic boundary condition, with
the interior and exterior dielectric constants set as 2 and 80, respectively, and assuming an ionic strength corresponding to a 0.145 mol/L salt solution. The solvent accessible surface of the molecule was
displayed using the Connolly algorithm with a solvent probe radius of
0.14 nm. This surface was colored red for potentials less than  4
kT (acidic), blue for potentials greater than 4 kT (basic), and white
for neutral potentials of 0 kT. Linear interpolation was used to
produce the colors between these values.
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RESULTS |
Homology models for the vWF-A1, -A2, and -A3 domains.
The homology modeling of the vWF-A1, -A2, and -A3 structures on the
basis of five related crystal structures28-31 was
straightforward (Fig 1). The use of a multiple sequence alignment for
the vWF-A superfamily significantly enhanced the reliability of
aligning the vWF-A1, -A2, and -A3 sequences with those of the crystal
structures.25 The three vWF-A structures containing 172 to
182 residues were modeled starting from the CR3 crystal structure using
five to six short remodeled loops with lengths of three to eight
residues that totalled 26 to 34 residues. Regions of sequence insertion and deletion corresponded to surface locations in the structure between
-helices and/or -strands, including the partial
exceptions of -helices A5 and A6. An additional three to four
residues were added at position 142/143 in the vWF-A1 domain and
position 92/93 in the vWF-A3 domain. Two segments of 3/4 residues and 6 residues were deleted at positions 109 to 112 and 157/158,
respectively, in the models. These changes showed that only minimal
perturbations of secondary structures were required in the three
modelings, although a one-to-one correspondence of residues cannot be
assumed in the loop regions that were rebuilt.
Support for a satisfactory outcome of the modelings was provided by
DSSP analysis of the secondary structure to show that all 13 -helices and -strands seen in the crystal structures were
retained after energy refinement, except for the truncated A5 -helix
in the vWF-A2 domain (Fig 1). Likewise the COMPARER analysis of
sidechain accessibilities in the three models showed that these agreed
with the crystallographic values (Fig 1). Two glycosylation sites in
the vWF-A2 domain at N751 on -helix A1 and N811 between -helices
A3 and A4 were exposed to solvent as required. All three vWF-A models
also satisfied stereochemical verification by PROCHECK.
The use of the vWF-A1 and -A2 models was enhanced by the use of 28 mammalian vWF-A1 and -A2 sequences representing at least 15 placental
orders and mostly derived from the recent sequencing of exon 28 in the
vWF gene.32 Their alignment
(Fig 2)
showed only a single one-residue insertion in the A1 sequences and none in the A2 sequences, and no gaps. On the basis of Fig 2, residues that
are seen to be 100% conserved in all 28 sequences may be essential for
function or structural folding in these two domains.
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| Fig 2.
Alignment of vWF-A1and vWF-A2 sequences from 28 mammalian species. The sequences are identified by accession numbers.
The numbering is taken from human vWF, and the location of -helices and -strands follows that of Fig 1. Residues that are 100%
absolutely or conservatively conserved are indicated by an asterisk.
(Conservative: G=A=S, D=E, I=L=V=M; S=T, R=K=H,
F=Y=W=H; X is disregarded). (A) The positions of D520 and E626 at
the active site and the proposed heparin site RK-RR-K (residues
571-585) are marked by a dollar sign ($). The positions of the type 2B
and 2M mutations are marked by a number sign (#). (B) The position of
D744 at the active site and the protease cleavage site at Y842-M843 are
marked by $. The positions of the type 2A mutations are marked by #.
The positions of two conserved putative N-linked glycosylation sites
are marked by N.
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Structure and function of the vWF-A1 domain.
The vWF-A protein fold is closely related in topology to the
doubly-wound / fold found in intracellular nucleotide binding proteins such as flavodoxin and the GTP-binding domain of
ras-p21.26,27 In the latter, the nucleotide binding site
occurs on the carboxy-edge of the parallel -sheet found at the
center of the fold, close to the switch point when the -strands wind
in opposite directions, and this creates a crevice that is where the
active site is located39 (Fig
3). The -helices A4, A5, and A6 are above the plane of the -sheet
to the right as visualized in Fig 3, whereas the -helices A1, A2,
and A3 to the left are below this plane. In many vWF-A domains, two
buried conserved Asp residues at positions 21 and 127 in Fig 1 are
located at this switch point, which serves as an active site and binds
metal.25,26 The CR3 and LFA-1 crystal structures show bound
metal at this location.28-31
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| Fig 3.
Supersecondary structure topology for the doubly wound
open / fold of the vWf-A domain. -Helices are represented as
cylinders, for which those above the central -sheet are shaded and
those below are unshaded. The -strands are shown as arrows and
labeled to follow Fig 1. N and C denote the N- and C-terminus,
respectively. The positions of mutations are shown on the appropriate
topology diagram as filled circles. Residues important in ristocetin
binding are shown as clear circles in the vWF-A1 domain. Residues
important in the protease cleavage site Y842-M843 are likewise shown as clear circles in the vWF-A2 domain.
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Inspection of the vWF-A1 domain shows that D520 is located in this
active site crevice and is fully conserved in CR3, LFA-1, and other
vWF-A sequences (Fig 1) and in all the 28 vWF-A1 sequences (Fig 2).
However, the second Asp residue at position 127 in CR3 and LFA-1 (Fig
1) is missing from all 28 vWF-A1 sequences. Although the conserved
vWF-A1 residue E626 is close to this position, this omission implies
that no metal binding occurs at this location in vWF-A1. Residues
important for the ristocetin-induced binding of vWF to platelets have
been shown to be D520-R524, E527-E531, K534, and E626 in vWF-A1 by
scanning alanine mutagenesis.40 Figure 2 shows that D520,
S522, E529, F530, K534, and E626 are conserved in vWF from 28 species.
As ristocetin is cationic, the involvement of D520, E529, and E626 is
of great interest, and the similar location of the ristocetin and
Mg2+ coordination sites in the vWF-A structure is shown in
Fig 4. Because Fig 3 shows that these three acidic
residues are close to the active site, it is concluded that the crevice
is of functional significance in the vWF-A1 domain.
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| Fig 4.
Comparison of the active sites in the vWF-A1 and vWF-A2
domains. The upper panels compare the ristocetin-binding residues in
the vWF-A1 domain with the Mg2+ binding site in the CR3
vWF-A domain. These residues are shown as dot representation of the van
der Waal radii of the residues in question. For comparison with Fig 6,
the type 2M and 2B mutation sites in the vWF-A1 domain are indicated
using spheres at the -carbon positions. The lower panels show the
proposed heparin binding site on the -helices A3 and A4 in the
vWF-A1 domain, and the protease cleavage site at Y842-M843 on the
vWF-A2 domain, also using -carbon positions.
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The heparin binding site in the vWF-A1 domain can be predicted from the
model. This has previously been mapped to residues 565 to 587, and the
binding of sulphatides has likewise been mapped to residues 569 to
584.2,41 Both ligands are negatively charged and are
complementary in charge to the positively charged surface seen in
electrostatic maps at this region of the vWF-A1 model (Fig 5). Within this sequence, Fig 2 shows
that the R571-K572 pair, the R578-R579 pair, and K585 are fully
conserved in 28 species and that R573 is conserved in 24 species. The
significance of this observation is underscored by noting that this
pattern of three sets of conserved basic residues is not found in any
of the other 72 vWF-A domains in the sequence alignment for this superfamily.25 The vWF-A1 model shows that these five basic residues are located on the -helices A2 and A3 in an extended linear
structure (Fig 4). The K572-R573 pair, the R578-R579 pair, and K585
show 60% to 100% sidechain accessibility to solvent and to heparin
(Fig 1). In addition, the -carbon distances between adjacent sets of
these residues are between 10 to 14 Å and are maximally 22 Å between
the first and last ones. These dimensions are comparable with those
seen for two known heparin binding sites. In the crystal structure of
antithrombin III (Brookhaven code 1ant), a heparin binding site has
been located on an extended region that involves five basic residues
R24, K114, R47, K125, and R129.42 The distances between
adjacent pairs of these -carbon atoms range between 6 to 10 Å, and
the distance between the first and fifth -carbon atoms spans 26 Å.
In the crystal structure of the basic fibroblast growth factor-heparin
complex (codes 1bfb and 1bfc), heparin makes contact with the basic
residues K27, R121, K126, and K136 (whose -carbon atoms are arranged
as a near-rectangle on loops between -strands at one end of the
protein), and no conformational changes were observed between the
heparin-free and heparin-bound structures.33 The distances
between adjacent pairs of -carbon atoms of these four basic residues
are between 6 to 9 Å and are maximally 14 Å between any pair.
Although it is not clear whether all three residues within the set
R571-K572-R573 are involved in heparin binding in vWF-A1, it is
concluded that the accessibility and separation of these three sets of
basic residues in vWF-A1 is comparable with the heparin-binding
residues in antithrombin III and basic fibroblast growth factor.
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| Fig 5.
Electrostatic maps of the vWF-A domains in human vWF. The
electostatic maps show the Connolly surface of the domains with a color
range of red representing a potential of less than 4 kT, blue a
potential of more than +4kT (basic) and white as 0kT (neutral). Linear
interpolation of the colors represents potentials between 4 kT and
+4 kT. The electrostatic surfaces are shown alongside ribbon diagrams
of the protein backbone of the respective domains. The domains are
shown in two perspectives, rotated along the vertical axis by 180°.
The ribbon views permit comparison with Figs 4 and 6.
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Heparin corresponds to a disaccharide repeat of L-iduronic acid and
D-glucosamine with three types of sulphate groups connected to the
sugar rings via an O-link, a CH2-link, and an N-link. The sulphate and carboxyl groups are positioned on the surface of a central
ribbon of sugar rings.33,43 The distances between neighboring pairs of the same type of sulphate group in heparin is 12.5 Å from one side of the ribbon to the opposite and 17.5 Å on the same
side of the ribbon (codes 1bfb, 1bfc, and 1hpn). The shortest distances
between different types of sulphate groups on the same side of the
ribbon are 6 to 8 Å. In relation to the predicted heparin binding
site, the separations of anionic groups in heparin are compatible with
those found for the conserved residues R571-K572, R578-R579, and K585
in the vWF-A1 domain.
The binding of the snake venom botrocetin to vWF-A1 occurs at different
locations to that of ristocetin.5 Peptide studies have
shown that residues 539-553, 569-583, and 629-643 are critical in
mediating botrocetin-induced vWF binding to platelets. The first two of
these correspond to the heparin binding region, whereas the third is
close to the ristocetin binding residues on the opposite face of the
vWF-A1 structure (Fig 3). As botrocetin is a large protein, its size
may either permit it to mask a large surface of the vWF-A1 domain or to
induce conformational changes that are transmitted through the vWF-A1
domain to affect both sides of the domain.
Mapping of type 2B and 2M vWD mutation sites in the vWF-A1
domain.
The mapping of eight type 2B vWD mutation sites in the vWF-A1 domain
(Table 1) showed that these are located in one spatial region of the
structure (Figs 3 and 6). This
is at the amino-edge of the -sheet, and all are located below the
plane of this -sheet. Four mutations (R543W, R545C, W550C, and
P574L) correspond to replacements of surface-exposed loop residues that
are moderately conserved in vWF from 28 species. Two mutations, V551L/F
and V553M, correspond to the replacement of two fully conserved smaller
hydrophobic residues by larger ones in the buried -strand BB (Fig
2a). Both these are presumed to disrupt the packing of the local
protein structure. The insertion of a third methionine at 540insM
(where M540 and M451 are both buried) will alter the conformation and packing of -helix A1. One mutation involves the replacement of the
predicted heparin-binding residue R578Q with a neutral residue. The
location of these mutations in the vWF-A1 model corresponds well to a
region that is situated between the heparin site at -helices A2 and
A3 and the GpIb binding site between residues 514-542 that encompasses
-strand BA and -helix A1. As the effect of heparin binding is to
inhibit vWF,41 it is concluded that the type 2B mutations
(while not necessarily preventing heparin binding) will perturb the
local protein structure involved in heparin binding and the
transmission of an allosteric signal from the heparin site to the GpIb
site. This would explain why these modifications upregulate vWF binding
to GpIb.
The mapping of the type 2M mutation sites showed that these are
distributed on the carboxyl-edge and amino-edge of the -sheet, and
all are above the plane of the -sheet (Fig 3). This occurred in a
different region of vWF-A1 from that corresponding to the type 2B
mutations. The three single-site 2M mutations (G561S, R611H/C, and
K645del) involve high solvent accessibilities and are located on loops.
As two of these three residues are fully conserved in all species,
whereas the third K645del corresponds to a deletion of one of four
lysines of which K642 and K643 are fully conserved, this implies that
loop structures have been perturbed. Because type 2M vWD corresponds to
a downregulation of vWF binding to GpIb, it is possible that the GpIb
binding site is directly affected. This could be confirmed in the case
of R611, because R611 is close to D514 at the N-terminus of the vWF-A1
domain and D514 has been identified to be important for the interaction
of vWF with platelets via the GpIb receptor.44 Both R611
and D514 are fully conserved in all 28 vWF-A1 sequences (Fig 2), and
D514 is unusual in that it is buried (Fig 1). Molecular graphic views show that these form a well-defined salt bridge in the crystal structure of CR3 (Fig 3). The multiple-residue 2M Milwaukee deletion R629-Q639 is at the loop between BD and A5 (Fig 2). The construction of
a vWF-A1 model with this deletion was stereochemically feasible, and no
loss of secondary structure was found apart from a shortening of the
-helix A5 by four residues. The effect of this mutation is
nonetheless sizeable, and would be expected to alter the protein structure at its GpIb binding site between residues 514 and 542 (Fig
3). Although there is no simple explanation for the effect of the
remaining mutations at G561 and K645 at present, it is conceivable that
they, too, can influence GpIb binding.
Structure and function of the vWF-A2 domain.
The main structural difference between the vWF-A2 and -A1 models is
that the former has a more even distribution of positive and negative
charges on its surface than the predominantly positively charged
surface of the latter (Fig 5). The vWF-A2 domain is the only one of the
three in vWF that contains two putative glycosylation sites at N752 and
N811. Figure 2 shows that these are fully conserved in all 28 vWF-A2
sequences, suggesting that this may be important.
A metalloprotease that is involved in the physiological cleavage of
large vWF multimers acts on the vWF-A2 domain.6,7 In the
presence of divalent cations with Ca2+ as the
activator,45 it cleaves the peptide bond between Y842 and
M843. Both Y842 and M843 are fully conserved in the 28 vWF-A2 sequences. In addition, M843 corresponds precisely in position to the
absent second conserved Asp residue in CR3 and LFA-1 that binds
Mg2+ in most members of the vWF-A superfamily and,
therefore, is located centrally in the active site of the doubly-wound
/ fold.39 The vWF-A2 model also shows that the two
conserved glycosylation sites N752 and N811 are close to the active
site but on the opposite side of this crevice from Y842 and M843 (Fig
4). It is possible that the glycosylation of these sites may define
access to this region to direct protease activity specifically towards
Y842 and M843.
Whereas not all type 2A mutations are localized to the vWF-A2 domain,
those within this domain (Table 1) are found in diverse locations (Figs
3 and 6). All 13 mutations correspond to fully conserved residues in
vWF from 28 species, except that the L817P mutation corresponds to
L817, which is conserved in 27 of the 28 species (Fig 2b).
Interestingly, the sidechain solvent accessibilities of the 13 mutation
sites (Fig 1) showed that nine correspond to buried locations, unlike
the predominantly surface-exposed mutation sites in types 2B and 2M
vWD. These are G742E/R, S743L, F751C, L777P, L817P, R834W/Q, V844D,
I865T, and E875K. Three more surface-exposed 2A mutations (L799P,
S850P, and P885S) involved mutations to or from Pro residues, which are
sterically constrained in their sidechain conformations, and one
(G846R) involved a mutation from a small residue to a larger one.
There are two groups of type 2A mutations.11,20 The group I
mutations are characterized by abnormal intracellular transport resulting in a decrease in vWF secretion and a selective loss of the
larger vWF multimers, and this may be consistent with defective protein
folding. The group I mutation sites known to date (G742E/R, S743L,
L777P, L817P, and V844D) are not spatially located to one region in
vWF-A2 (Figs 2 and 3). The group II mutation sites (G742E/R, L799P,
I865T, and R834W/Q) are characterized by the secretion of normal vWF
multimers but are sensitive to protease activity. These sites are also
not located spatially in vWF-A2. One site at F751C is adjacent to the
conserved glycosylation site at N752. Because all these mutations
correspond to conserved buried or sterically constrained residues, this
implies that type 2A vWD is associated with alterations in protein
folding, which, although minor in scope, are sufficient to disable its
secretion or increase its susceptibility to proteolysis.
Structural properties of the vWF-A3 domain.
Although the protein model of the vWF-A3 domain is similar to those of
the -A1 and -A2 domains, its electrostatic map shows that there is a
large negatively charged area on its front surface that is not seen in
the -A1 and -A2 domains, and this corresponds to the crevice of the
doubly-wound / fold. No large positively charged areas were found
that correspond to those seen in the vWF-A1 domain. The negatively
charged surface of the A3 domain resembles one calculated for the
corresponding area of CR3 (not shown) where Mg2+ is
coordinated. The DxSxS motif of half the metal binding site of CR3 at
positions 21 to 25 in Fig 1 is conserved in the vWF-A3 domain, unlike
the vWF-A1 and -A2 domains. Although two Asp residues are found at
positions 94 and 128 in the vWF-A3 domain that correspond to the other
half of the CR3 metal-binding motif at residues 94, 127 ,and 129, the
latter three metal binding residues are not conserved in vWF-A3. It is
unlikely that the active site of this domain interacts with cations.
The vWF-A3 domain is critical for the binding of vWF to
collagen,9,10 and residues 948 to 998 have been implicated
in this.2 These residues are mapped to the N-terminal
region of the domain including the -helices A1 to A3 below the
central -sheet. The molecular mechanism of the interaction with
collagen is unknown. The binding of some collagen groups to platelets
is often mediated and dependent via divalent cations, in which the binding of collagen types I, III, and IV to platelet receptors at high
shear stress rates is particularly sensitive to variations in
Mg2+ concentration.46 Possibly the binding of
vWF-A3 to collagen acts by a similar mechanism.
| |
DISCUSSION |
Past experience with homology modeling47,48 shows that,
although the accuracy of the models will be better in the protein core
where it is aligned with the 13 -helices and -strands and will be
reduced in the rebuilt loop regions (Fig 1), the use of a good
alignment from multiple sequences25 together with known homologous crystal structures28-31 is sufficiently accurate
to assess vWF function.
Functional sites in the three vWF-A domains.
Residues at the switch point between the two halves of the -sheet in
the vWF-A domains have functional significance because these occur in a
crevice. The conserved DxSxS motif and the Thr, Asp, and Glu residues
in the metal binding site of CR3 is conserved in many vWF type-A
domains. Despite that, this is only partially conserved in vWF-A3, and
is replaced in vWF-A1 by DxSxR and in vWF-A2 by ExSxK. Metal binding is
not predicted for the vWF-A domains of vWF. Despite these sequence
differences, the present modeling has shown that this active site
region in vWF-A is involved in ristocetin binding in vWF-A1 and
contains the physiologically relevant protease cleavage site in vWF-A2.
In distinction to these active sites, the predicted heparin binding
site R571-K572-R578-R579-K585 occurs on the reverse side of the vWF-A1
structure to that of the active site. An analogous location was
identified for the heparin binding site in antithrombin III, which is
also distant from the physiological active site in this
protein.42
Mutation sites on the vWF-A1 domain.
Whereas earlier analyses of the type 2B mutation sites on the vWF-A1
domain had indicated some degree of spatial
clustering,26,30 the present modeling now showed that all
the currently known type 2B mutations are grouped at the amino edge of
the -sheet in the vWF-A1 domain below the -sheet and close to a
predicted heparin binding site. In distinction, the type 2M mutations
correspond to a spatially different set of residues that are located on
the carboxyl-edge of the -sheet above the -sheet (Fig 6). It is likely that the separate location of these mutation types in vWF-A1 are
related to the upregulation (type 2B) or downregulation (type 2M) of
GpIb binding by vWF. It is known that heparin binding inhibits the
vWF-GpIb interaction.49 The simplest molecular
explanation of these mutations is that type 2B vWD involves the
decoupling of the inhibitory effects of heparin binding on the vWF-GpIb
interaction, leading to upregulation, whereas type 2M involves the
disabling of GpIb binding to vWF, leading to downregulation. Both these explanations require the involvement of two vWF-A1 conformations that
are transmitted through its structure. This is consistent with a recent
proposal that the vWF-A1 domain exists in either the "on" or
"off" states,15 and that the type 2B mutations switch it to "on" to facilitate GpIb binding. The ristocetin-binding site would be left exposed to facilitate vWF binding to GpIb. Evidence
that other members of the vWF-A superfamily can transmit such signals
has been observed in LFA-1. Mutations at residues IKGN at the
N-terminus of LFA-1 (Fig 1), which is at the opposite end of the vWF-A
structure to the metal-binding site (Fig 3), disrupt the interactions
of LFA-1 with its ligand intracellular adhesion molecule-3 (ICAM-3)
without affecting the interactions of LFA-1 with
ICAM-1.31,50 Structural studies on the LFA-1 vWF-A domain
show that there are significant alterations in the metal binding site
in the bound and free states.28 In distinction to this, but
consistent with this, the structure of the CR3 vWF-A domain has been
seen to switch between two conformations depending on whether
Mg2+ or Mn2+ is bound.28,29
The type 2M mutations are characterized by decreased vWF-A1 binding to
GpIb, as detected by reduced vWF binding to platelets in the presence
of ristocetin, as well as from binding studies of recombinant wild-type
and mutated vWF-A1 to GpIb. This is often accompanied by a decreased
vWF binding to platelets in the presence of botrocetin. These
observations imply that the molecular effect of the type 2M mutations
would involve the direct perturbation of the ristocetin or GpIb binding
sites. Evidence for this was found from the analysis of the type 2M
mutation involving the loss of R611, which in turn causes the salt
bridge with D514 in the GpIb binding site to be lost. Likewise the
large deletion R629-Q639 would be expected to perturb GpIb binding
directly.
Mutation sites on the vWF-A2 domain.
Defective mutations in the vWF-A2 domain do not involve direct
functional interactions with ligands. Unlike the type 2B and 2M
mutations, all the type 2A mutations were found to involve sterically-significant residues that were fully conserved at buried sites in vWF sequences from 28 species. This implies that the mutations
may correspond to a modified folding of the vWF-A2 protein structure,
which would affect its physiological function directly or indirectly
through a signal transmission mechanism. Several of the type 2A
mutations are close to the protease cleavage site at Y842-M843 or to
the nearby glycosylation site at N752. Evidence that makes this
hypothesis likely is found from the nature of groups I and II of type
2A vWD, which are implicated in the secretion of vWF and its stability
to proteolytic attack. An altered folding of the vWF-A2 domain may
hinder vWF secretion or alter the likelihood of physiological cleavage.
| |
CONCLUSION |
In this study, the comparative homology modeling of the vWF-A1, -A2,
and -A3 domains has been successful in correlating known biochemical
properties of these three domains with the basic molecular features of
the / fold in these structures. Although the models will not
substitute for the atomic detail available in crystal structures for
each domain, recent reports of forthcoming crystal structures for the
vWF-A1 and -A3 domains support the use of homology modeling for these
structures.51,52 In the present study, the active site
crevices in the vWF-A1 and -A2 domains were shown to contain
functionally important residues, and a separate heparin binding site in
the vWF-A1 domain was predicted. The models have also enabled a
molecular mechanism for the vWD type 2B, 2M, and 2A mutation sites to
be assessed. This explanation of the molecular defects arising from
these mutations will facilitate the rational planning of experiments to
explore the molecular pathology of vWD. In addition, the sequence
conservation analysis of Fig 2 together with the views in Figs 3 and 6
will provide a rational basis for the assessment of further mutation
sites that may be discovered. This is exemplified by our recent
characterization of the K645del mutation and its identification as a
type 2M defect.18,53
| |
FOOTNOTES |
Submitted May 20, 1997;
accepted November 6, 1997.
Address reprint requests to Stephen J. Perkins, DPhil,
Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, Rowland Hill St, London NW3 2PF, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
P.V.J. thanks members of the Protein Structure Unit in the Department
of Biochemistry and Molecular Biology for their assistance and helpful
advice. S.J.P. thanks the Wellcome Trust for financial support.
| |
REFERENCES |
1.
Ruggeri ZM:
von Willebrand factor.
J Clin Invest
99:559,
1997[Medline]
[Order article via Infotrieve]
2.
Meyer D,
Girma J-P:
von Willebrand factor: Structure and function.
Thromb Haemost
70:99,
1993[Medline]
[Order article via Infotrieve]
3. Ruggeri ZM, Ware J: von Willebrand factor. FASEB J 7:308, 1993
4.
Sadler JE:
von Willebrand factor.
J Biol Chem
266:22777,
1991[Free Full Text]
5.
Sugimoto M,
Mohri H,
McClintock RA,
Ruggeri ZM:
Identification of discontinuous von Willebrand factor sequences involved in complex formation with botrocetin.
J Biol Chem
266:18172,
1991[Abstract/Free Full Text]
6.
Dent JA,
Berkowitz SD,
Ware J,
Kasper CK,
Ruggeri ZM:
Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor.
Proc Natl Acad Sci USA
87:6306,
1990[Abstract/Free Full Text]
7.
Furlan M,
Robles R,
Lämmle B:
Partial purification of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis.
Blood
86:4223,
1996
8.
Shelton-Inloes BB,
Titani K,
Sadler JE:
cDNA sequences for human von Willebrand factor reveal five types of repeated domain and five possible protein sequence polymorphisms.
Biochemistry
25:3164,
1986[Medline]
[Order article via Infotrieve]
9.
Cruz MA,
Yuan H,
Lee JR,
Wise RJ,
Hardin RI:
Interaction of the von Willebrand factor (vWF) with collagen.
J Biol Chem
270:10822,
1995[Abstract/Free Full Text]
10.
Lankhof H,
van Hoeij M,
Schiphorst ME,
Bracke M,
Wu Y-P,
Ijsseldijk JW,
Vink T,
de Groot PG,
Sixma JJ:
A3 domain is essential for interaction of von Willebrand factor with collagen type III.
Thromb Haemost
75:950,
1996[Medline]
[Order article via Infotrieve]
11.
Ginsburg D,
Sadler JE:
von Willebrand disease: A database of point mutations, insertions and deletions.
Thromb Haemost
69:177,
1993[Medline]
[Order article via Infotrieve]
12.
Sadler JE,
Matsushita T,
Dong Z,
Tuley EA,
Westfield LA:
Molecular mechanism and classification of von Willebrand disease.
Thromb Haemost
74:161,
1995[Medline]
[Order article via Infotrieve]
13.
Eikenboom JC,
Reitsma PH,
Briët E:
The inheritance and molecular genetics of von Willebrand's disease.
Haemophilia
1:77,
1995
14.
Ribba AS,
Christophe O,
Derlon A,
Cherel G,
Siguret V,
Laverge JM,
Girma JP,
Meyer D,
Pietu G:
Discrepancy between IIA phenotype and IIB genotype in a patient with a variant of von Willebrand disease.
Blood
83:833,
1994[Abstract/Free Full Text]
15.
Cooney KA,
Ginsburg D:
Comparative analysis of type 2b von Willebrand disease mutations: Implications for the mechanism of von Willebrand factor binding to platelets.
Blood
87:2322,
1996[Abstract/Free Full Text]
16.
Rabinowitz I,
Tuley EA,
Mancuso DJ,
Randi AM,
Firkin BG,
Howard MA,
Sadler JE:
A missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein IB.
Proc Natl Acad Sci USA
89:9846,
1992[Abstract/Free Full Text]
17.
Hilbert L,
Gaucher C,
Mazurier C:
Identification of two mutations (Arg611Cys and Arg611His) in the A1 loop of von Willebrand factor (vWF) responsible for type 2 vo |
Abstract
Introduction
Abstract