Recombinant a2-E318W VWFA domain showed elevated and specific binding to collagen I compared with the wild-type.. The E318W mutation had additional effects on VWFA domain properties as a
Trang 1A novel gain-of-function mutation of the integrin a2 VWFA domain
Alexis Aquilina1, Michelle Korda1, Jeffrey M Bergelson2, Martin J Humphries1, Richard W Farndale3 and Danny Tuckwell1
1 School of Biological Sciences, University of Manchester, Manchester, UK; 2 The Children’s Hospital of Philadelphia, Philadelphia,
PA, USA; 3 Department of Biochemistry, University of Cambridge, Cambridge, UK
Integrin a2b1 is the major receptor for collagens in human
tissues, being involved in cell adhesion and the control of
collagen and collagenase gene expression The collagen
binding site of a2b1 has been localized to the a2 von
Wille-brand Factor type A (VWFA) domain (A-domain or
I-domain) and the residues responsible for the interaction
with collagen have been mapped We report a study of a2
VWFA domain in which residue E318, which lies outside the
collagen binding site, is mutated to tryptophan, showing that
this is a gain-of-function mutation Recombinant a2-E318W
VWFA domain showed elevated and specific binding to
collagen I compared with the wild-type Side chain
hydro-phobicity was important for the gain-of-function as elevated
binding was seen with E318I and E318Y, but not with
E318R The E318W mutation had additional effects on
VWFA domain properties as a2-E318W VWFA domain
differed from the wild-type in its cation preferences for ligand binding and in binding to monoclonal antibody JA203, which bound at a site distal to E318 The gain-of-function effect was not restricted to binding to collagen I as a2-E318W also showed elevated binding to collagen IV, collagen I C-propeptide, laminin and E-cadherin Binding to these ligands was inhibited by collagen peptide containing the GFOGER motif, indicating that these bound to the VWFA domain by a similar mechanism to collagen I These data indicate that residue E318 plays a novel and important role in modulating a2 VWFA domain–ligand binding and may be involved in the conformational changes associated with its regulation
Keywords: adhesion; collagen; extracellular matrix; integrin
Integrin a2b1 is the major human collagen receptor,
expressed on a wide range of cell types in vivo [1] It has
been shown to mediate cell adhesion in vitro to a range of
collagens [2–4], but is also a receptor for a number of
noncollagenous molecules including laminins, collagen
C-propeptides, E-cadherin, and certain viruses and snake
toxins [5–10] (J Whittard, A P Mould, A Koch, O Pertz,
J Engel, M J Humphries, unpublished data) Binding of
a2b1 to collagen induces collagen and collagenase gene
expression [11,12] and the initiation of the p38 MAPK
signalling pathway [4] a2b1 is also responsible for force
generation by cells in collagen gels [13] and may be involved
in matrix assembly [14,15] In vivo, a2b1 plays an important
role in platelet adhesion to collagens during thrombus
formation [16], and although a2b1 is probably not the
major collagen receptor on platelets [17], a genetic
predis-position to increased levels of platelet a2b1 may be a risk
factor for stroke [18], myocardial infarction [19], and
diabetic retinopathy [20] (although see [21,22]) a2b1 has
also been found to be involved in the regulation of
inflamatory responses in experimental models of hypersen-sitivity and arthritis [23]
The ligand binding site of a2b1 is located in the 200 amino acid von Willebrand Factor type A domain (VWFA domain, also known as the A- or I-domain) of the a2 subunit The a2 VWFA domain can be produced as a recombinant protein, which reproduces the ligand specifi-city, affinity and cation preferences of the complete molecule [8,24–29] The recognition sequence for a2 VWFA domain
on collagen I has been identified and contains an essential GER sequence [30] The determination of the structure of a2 VWFA domain complexed with the collagen peptide demonstrates that the E of the peptide coordinates with a cation bound to the VWFA domain, forming a metal-ion-dependent adhesion site (MIDAS) [31,32] Recognition sequences for the other noncollagenous ligands of a2b1 have yet to be determined
Comparison of the structure of the a2 VWFA domain complexed with collagen (ÔopenÕ conformation) and alone (ÔclosedÕ conformation) [31,32] indicates that collagen bind-ing is accompanied by important conformational changes in the a2 VWFA domain, in particular in the C-terminal helix a7 A number of studies indicate that similar conforma-tional changes in the aM and aL VWFA domains accompany ligand binding [33–35] In aM, residue F302, which lies at the N-terminal end of helix a7, is buried in the closed conformation, but exposed in the open form The substitution of a tryptophan at position F302 in aM led to
an increase in ligand binding, increased binding of an antibody associated with aMb2 activation, and an increase
in the proportion of active integrin [36] This indicates that F302 plays an important role in modulating ligand binding,
Correspondence to D Tuckwell, 2.205 Stopford Building, School of
Biological Sciences, University of Manchester, Oxford Road,
Manchester, M13 9PT, UK Fax: + 44 161 275 5082,
Tel.: + 44 161 275 5061, E-mail: danny.tuckwell@man.ac.uk
Abbreviations: GST, glutathione S-transferase; MIDAS, metal
ion-dependent adhesion site; VWFA domain, von Willebrand factor type
A-domain.
(Received 17 September 2001, revised 26 November 2001, accepted
14 December 2001)
Trang 2possibly by promoting the open conformation, although
this is debated [37]
Here we report an investigation into the function of the
residue corresponding to aM F302 in the a2 VWFA
domain, residue E318 Although E318 lies outside the
collagen binding site, introduction of the mutation E318W
resulted in increased ligand binding, changes in cation
preferences and altered antibody epitope expression We
also examined the molecular basis for the gain-of-function
and exploited this property to study a2 VWFA domain
interactions with noncollagenous ligands of a2b1 These
data indicate that E318W has a similar effect on a2 as F302
does on aM and that residue E318 has an important role in
modulating a2 VWFA domain function
M A T E R I A L S A N D M E T H O D S
General reagents
Acid soluble rat tail type I collagen and EHS-laminin were
obtained from Sigma-Aldrich Collagen peptides were
pre-pared as described in [30] Collagen IV fragment CB3 was the
kind gift of K Ku¨hn, Max-Planck-Institute for
Biochemist-ry, Martinsreid, Germany [38]; collagen C-propeptide was
prepared as described in [7,8]; the E-cadherin-COMP
construct comprising the five extracellular domains of mouse
E-cadherin fused to the assembly domain of rat cartilage
oligomeric matrix protein (COMP) was the kind gift of
J Engel, Bı´ozentrum, University of Basel, Switzerland
Mutagenesis and production of recombinant VWFA
domains
The generation of the wild-type a2 VWFA domain
construct has been described previously [26] Mutagenesis
of this construct was carried out using the mutagenesis
protocols described by Kunkel [39] and the BIO-RAD
Muta-Gene mutagenesis kit Essentially, single-stranded
uracil-containing DNA was generated by helper phage
infection of Escherichia coli strain CJ236 and then used as
the template for in vitro second-strand syntheses with the
following mutagenic oligonucleotides (bases differing from
the wild-type sequence are underlined): for E318 to W,
5¢-TTCAATGTGTCTGATTGGGCAGCTCTACTAGA
AAAGGCTG-3¢; for E318 to I, 5¢-CAATGTGTCTGA
TATAGCAGCTCTACTAGAAAAG-3¢; for E318 to R,
5¢-CAATGTGTCTGATCGAGCAGCTCTACTAGAAA
AG-3¢; for E318 to Y, 5¢-CAATGTGTCTGATTATGCA
double-stranded DNA was used to transfect E coli strain DH5aF¢,
and single colonies containing the mutant DNA were
identified by DNA sequencing Wild-type and mutant
recombinant VWFA domain–glutathione S-transferase
(GST) fusion proteins were produced and purified as
described previously [9,26,27]
Binding assays
Solid phase binding assays to measure binding of
biotiny-lated collagen to a2 vWFA-domain were carried out as
follows: Immulon 4 microtitre plates (Dynex, Billinghurst,
West Sussex, UK) were coated with 100 lL a2
vWFA-domain fusion proteins diluted in 136.8 mMNaCl, 8.1 mM
Na2HPO4, 2.7 mMKCl, 1.5 mMKH2PO4, 0.9 mMCaCl2, 0.5 mM MgCl2, pH 7.4 (NaCl/Pi+ where the + sign indicates the presence of Mg and Ca ions) overnight at
4°C The following day, protein solutions were removed and wells blocked with 200 lL 50 mgÆmL)1BSA, in 40 mM Tris/HCl, 150 mM NaCl, pH 7.4 (NaCl/Tris), for 2 h at room temperature Wells were then washed three times with 200 lL NaCl/Tris, 1 mgÆmL)1 BSA, 1 mM MgCl2 (buffer A), and to each well was then added 50 lL inhibitor (in buffer A at double the final concentration) followed by
50 lL biotinylated collagen I (also in buffer A at double the final concentration), for 3 h at room temperature Wells were then washed three times with 200 lL buffer A and
100 lL 1 : 200 (v/v) ExtrAvidin-peroxidase (Sigma) in buffer A added for 10 min at room temperature Wells were then washed three times with 200 lL buffer A, and colour developed by the addition of 2 mM 2¢2¢-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid), 0.03% (v/v) H2O2, 0.05M NaH2PO4, 0.1M sodium acetate, pH 5.0 (ABTS reagent) Absorbance was measured at 405 nm on a plate reader For experiments measuring collagen binding in the presence of different cation concentrations, the protocol was carried out as above up to the blocking stage, then wells were washed three times with 200 lL NaCl/Tris,
1 mgÆmL)1 BSA, then 50 lL cation (in NaCl/Tris,
1 mgÆmL)1 BSA at double the final concentration) was added followed by 50 lL biotinylated collagen I (also in NaCl/Tris, 1 mgÆmL)1BSA at double the final concentra-tion) All subsequent steps were as above
For the measurement of VWFA domain binding to immobilized collagen I, collagen IV CB3, C-propeptide, laminin, E-cadherin or polylysine, Immulon 4 microtitre plates (Dynex) were coated with 100 lL collagen I or other ligands diluted in NaCl/Pi+overnight at 4°C The follow-ing day, protein solutions were removed and wells blocked
as above Wells were then washed three times with buffer A, and to each well was added 50 lL inhibitor (in buffer A at double the final concentration) followed by 50 lL VWFA domain (also in buffer A at double the final concentration), for 3 h at room temperature Wells were then washed three times with 200 lL buffer A and 100 lL sheep anti-GST antiserum (the kind gift of V Allan and S Taylor, Univer-sity of Manchester, UK) 10 lgÆmL)1in buffer A added for
1 h at room temperature Wells were then washed three times with 200 lL buffer A and 100 lL peroxidase antisheep (DAKO), 1 : 1000 (v/v) in buffer A added for
1 h at room temperature Wells were then washed three times with 200 lL buffer A, and colour developed by the addition of ABTS reagent as above For assays measuring the binding of VWFA domain to collagen in the presence of increasing cation concentrations, NaCl/Tris was cleared of residual cations by the addition of 1 gÆL)1Chelex 100 overnight and the Chelex removed by filtration prior to use
in assays The assay protocol was carried out as above up to the blocking stage, then wells were washed three times with
200 lL NaCl/Tris, 1 mgÆmL)1BSA, and 50 lL cation (in NaCl/Tris, 1 mgÆmL)1BSA at double the final concentra-tion) was added followed by 50 lL VWFA domain (also in NaCl/Tris, 1 mgÆmL)1BSA at double the final concentra-tion) All subsequent steps were as above with the exception that buffer A contained 1 mMMgCl2and 1 mMMnCl2 Assays to measure antibody binding to recombinant VWFA domains were carried out after the method of
Trang 3Brookman et al [40] Immulon 4 microtitre plates were
coated with 100 lL 5 lgÆmL)1fusion protein in NaCl/Pi+,
overnight at 4°C Wells were then washed twice with
200 lL NaCl/Pi–(NaCl/Pi+without Ca2+or Mg2+) and
blocked with 100 lL 2% (w/v) fat-free milk powder, NaCl/
Pi–, for 1 h at 4°C Wells were then washed twice with
200 lL 0.1% (v/v) Tween 20, NaCl/Pi–, and 100 lL
antibody diluted in 0.5% (w/v) milk powder, 0.1% (v/v)
Tween 20, NaCl/Pi–, added for 2 h at 4°C Wells were then
washed twice with 200 lL 0.1% (v/v) Tween 20, NaCl/Pi–,
and peroxidase antimouse (for mouse monoclonals;
DAKO) or peroxidase antisheep (for sheep anti-GST)
diluted in 0.5% (w/v) milk powder, 0.1% (v/v) Tween 20,
NaCl/Pi–, added for 2 h at 4°C Wells were then washed
three times with 200 lL 0.1% (v/v) Tween 20, NaCl/Pi–, and
colour developed by the addition of ABTS reagent as above
R E S U L T S
The E318W mutation in a2 increases collagen binding
The introduction of the mutation F302W into aM has been
shown to result in a gain of function in both the isolated aM
VWFA domain and in aMb2 [36] Comparison of the a2
and aM sequences and structures indicated that the
homologous residue in a2 is E318 (Fig 1A), a residue in
the a7 helix which, like aM F302, undergoes a large
displacement on collagen binding and moves from a buried
to an exposed position (Fig 1B and C) a2 E318 was
therefore mutated to tryptophan, the recombinant mutant
VWFA domain generated, and the a2 E318W VWFA
domain tested in solid phase binding assays a2-E318W
VWFA domain showed enhanced binding to collagen I
compared with wild-type VWFA domain, both when binding of biotinylated collagen to immobilized VWFA domain (Fig 2A) and binding of VWFA domain to immobilized collagen was measured (Fig 2B) The inter-action of a2-E318W VWFA domain with collagen was specific as it could be inhibited by EDTA (Fig 2A and B) as well as by a collagen peptide containing the a2 recognition sequence GFOGER (Fig 2C) The data from the binding
of biotinylated collagen to VWFA domains were analysed
by curve fitting and double reciprocal plots (analyses were carried out on the results of four independent experiments): the apparent affinities for the wild-type and mutant VWFA domains were 3.3 lgÆmL)1and 0.5 lgÆmL)1, respectively, and the binding of wild-type VWFA domain, which is not saturated over the range shown in Fig 2A, was calculated
to reach 82% of the level of a2 E318W VWFA domain E318W therefore results in elevated collagen binding primarily by altering the apparent affinity of the VWFA domain–collagen interaction, although residue 318 does not form part of the collagen binding site (MIDAS) This increase in binding was not due to misfolding or differences
in stability of the VWFA domain as the previously characterized monoclonal antibodies JA202, JA208, JA215, JA218 and Gi9 [9] showed identical levels of binding
to wild-type and mutant VWFA domain (data not shown)
To determine the molecular basis of the effect of the E318W mutation, E318 was mutated to Y, I and R and recombinant VWFA domains tested: a2-E318Y and a2-E318I behaved similarly to a2-E318W, while a2-E318R showed reduced binding to collagen I compared with wild-type (Fig 3) Thus a hydrophobic residue at position E318 is required for the enhanced collagen I binding, but the size of the residue is less important The decreased binding of
Fig 1 E318 moves between the ‘open’ and ‘closed’ forms of a2 VWFA domain (A) Alignment of C-terminal sequence of a2 and aM VWFA domains showing a2 E318 and aM F302 (boxed) Secondary structural elements are marked above the alignment; dots indicate residues identical between a2 and aM (B, C) Structure of the C-terminal region of a2 VWFA domain, in the presence (B) or absence (C) of collagen peptide ligated The arrow indicates the position of the E318 side chain, which is altered by the conformational change.
Trang 4E318R relative to wild-type is likely to be due to effects on
global conformation, as this mutant was unstable and its
activity decreased over the course of a few days
The E318W mutation affects the MIDAS and helix a3 The interaction of collagens with the a2 VWFA domain requires the MIDAS cation and we have previously shown that either Mg2+or Mn2+will support VWFA domain– collagen binding [9,26] The effects of the E318W mutation
on the requirements for cations in collagen binding were therefore investigated The wild-type and mutant VWFA domains showed similar curves for binding of biotinylated collagen in the presence of Mg2+ but differed in their binding in the presence of Mn2+, with the a2-E318W VWFA binding curve shifted to the left compared with the wild-type, which displayed a complex profile (Fig 4A and B) Similar profiles for a2-E318W and wild-type were seen over a range of cation and fusion protein concentra-tions (data not shown) Assays in which the immobilized and soluble components were swapped (measuring the binding of VWFA domain to collagen-coated plates) also gave very similar profiles (Fig 4C,D) The a2-E318W mutation therefore affects the formation of the collagen– cation–VWFA domain complex at the MIDAS, leading to altered cation preferences compared with wild-type This is despite the mutation being topologically distinct from the MIDAS itself
In order to identify other regions which might be affected
by the E318W mutation, the panel of 21 anti-VWFA domain monoclonal antibodies, JA201–JA221 previously developed by us [9] was screened for differential binding to a2-E318W VWFA domain compared with wild-type Antibody JA203 was found to bind with a lower affinity
to a2-E318W VWFA domain than to wild-type (Fig 5A) Other antibodies bound to the wild-type and mutant domains at similar levels (data for JA202 is shown for comparison in Fig 5B) The epitope for JA203 was mapped using human–mouse chimeras which spanned the full extent
of the VWFA domain [41] This approach exploits the fact
Fig 2 a2-E318W shows elevated specific binding to collagen compared
with wild-type (A) Microtitre plates were coated with 10 lgÆmL)1
a2-E318W (n,m) or wild-type a2 (h,j) and the binding of biotinyated
collagen I measured in the presence of 1 m M MgCl 2 (m,j) or 1 m M
MgCl 2 /10 m M EDTA (n,h) Data are means ± SD; n ¼ 4 from two
experiments (B) Microtitre plates were coated with 1 lgÆmL)1
colla-gen I and binding of a2-E318W or wild-type a2 (2 lgÆmL)1) measured
in the presence of 1 m M MgCl 2 (dark bars) or 1 m M MgCl 2 /10 m M
EDTA (light bars) Data are means ± SD; n ¼ 7 from three
experi-ments (wild-type) and n ¼ 5 from two experiments (a2-E318W).
(C) Microtitre plates were coated with 1 lgÆmL)1collagen I and the
binding of 0.5 lgÆmL)1a2-E318W measured in the presence of 1 m M
MgCl 2 with the addition of 100 lgÆmL)1GFOGER collagen peptide
(which carries the a2 binding site), or control collagen peptide (which
does not carry the a2 binding site).
Fig 3 a2E318I and a2 E318Y show enhanced collagen I binding Binding of biotinylated collagen I to a2-E318 mutations E318W (r), E318I (m) E318Y (d) and E318R (s) is shown Binding to wild-type (j) and wild-type + EDTA (h) are shown for comparison Microtitre plates were coated with 10 lgÆmL)1VWFA domains and the binding
of biotinylated collagen I measured Binding of mutants in the pres-ence of EDTA was the same as that seen for wild-type + EDTA Data are means ± SD; n ¼ 6 from three experiments except for E318R where n ¼ 4 from two experiments.
Trang 5that JA203 was raised in mouse against a human antigen,
and will therefore bind to residues differing between human
and mouse a2 VWFA domain The binding of JA203 to
eight of the nine chimeras was similar to that of wild-type
VWFA domain, but no binding to the chimera covering
helix a3 was seen (Fig 6) The E318W mutation therefore
affects helix a3 Significantly, we have previously identified
this region as the binding site for other functionally relevant
antibodies [9] The helix a3 region is topologically distinct
from residue E318 and there is a number of chimeras which
alter amino acids located between E318 and helix a3 with no
effect on JA203 binding The differential binding of JA203 is
therefore not due to a mutation within its epitope The
cation binding and the JA203 data indicate that the E318W
mutation has specific effects on VWFA domain properties
in addition to collagen binding Because the sites affected
are topologically distinct from the site of the mutation, these
effects may be due to conformational changes which are
transmitted to the MIDAS and the JA203 epitope
The consequences of the E318W mutation for a2 function
a2b1 is a receptor not only for collagen I but also for
collagen IV, collagen I C-propeptide, laminin and
E-cad-herin [5,7–9,38], and so the effect of the E318W mutation on
the interaction of the a2 VWFA domain with other ligands
was investigated a2-E318W VWFA domain showed
elevated specific binding to the integrin-binding CB3
fragment of collagen IV and to all three noncollagen
proteins (Fig 7A) No similar increase in binding to the
control proteins, fibrinogen or the 50 kDa fragment of
fibronectin was seen This indicated that the enhancing
effect of the E318W mutation was not confined to collagens
alone Little is known about the molecular basis of a2
VWFA domain binding to its noncollagenous ligands and
the binding of wild-type a2 VWFA domain to laminin and
E-cadherin is typically much lower than to collagen I
However, the elevated binding seen with a2-E318W allowed
us to study these otherwise weak interactions Binding of
Fig 4 a2-E318W and wild-type a2 differ in their cation preferences for binding to collagen I (A, B) Microtitre plates were coated with 0.2 lgÆmL)1 a2-E318W (A) or 0.5 lgÆmL)1wild-type a2 (B) and the binding of 1 lgÆmL)1biotinylated collagen I measured in the presence of Mn2+(d), Mg2+ (m), or EDTA (e) (C, D) Microtitre plates were coated with collagen I (1 lgÆmL)1) and a2-E318W (C) or wild-type a2 (D) added at 1 lgÆmL)1in the presence of Mn2+(d) or Mg2+(m) and detected with anti-GST Ig Data are means ± range (n ¼ 2) from representative experiments.
Fig 5 JA203 shows differential binding to a2-E318W compared with wild-type a2 (A) JA203 binds with lower affinity to a2-E318W (s) than to wild-type a2 VWFA domain (d); (B) Binding of JA202 is identical for a2-E318W (s) and wild-type (d), and is shown for comparison Microtitre plates were coated with 5 lgÆmL)1VWFA domain and the binding of antibodies measured over a range of con-centrations Data are means±SD; n ¼ 6 from two experiments.
Trang 6a2-E318W to collagen IV CB3, C-propeptide, laminin and
E-cadherin was inhibited by EDTA (Fig 7A) and collagen
peptide (Fig 7B) Binding of a2-E318W to polylysine was
not inhibited These inhibitor studies showed that binding of
these ligands to a2 VWFA domain occurs at the same site as
collagen I and probably by the same mechanism
D I S C U S S I O N
We report a novel gain-of-function mutation of the a2
VWFA domain Introduction of the E318W mutation led
to increased specific collagen binding as well as alterations in
cation preferences for collagen binding and in the binding of
antibody JA203 These data suggested that the mutation
exerted its effect by inducing conformational changes in the VWFA domain We also describe further mutations of E318 which help to define the molecular basis of the gain-of-function effect as well as gain-of-functional studies showing that noncollagenous a2b1 ligands bind to the mutant VWFA domain at an elevated level relative to wild-type and by the same mechanism as collagen I The E318W mutation in a2 therefore has a similar effect as the equivalent mutation F302W in the aM VWFA domain, which also showed increased binding [36]
The molecular basis of the interaction between a2 VWFA domain and collagen I is now well understood following the solution of the X-ray crystal structure of the VWFA domain–collagen cocrystal [32] This interaction involves a discrete set of residues clustering round the cation binding site, as well as the cation itself It is of interest that the E318W mutation has such a large effect on the binding of collagenous and noncollagenous ligands but does not form part of the ligand binding site In addition to the effects on ligand binding, the mutation also affected the use of cations
by the ligand binding site Although the precise nature of the atomic events responsible for the shifts between preferences for Mg2+and Mn2+are not clear, changes in
Fig 6 The epitope for JA203 is located in the a3 helix The JA203 epitope was mapped using human-mouse a2 VWFA domain chimeras [41] The figure shows the mutations introduced to convert stretches of human to mouse sequence; the percentage binding of JA203, compared with wild-type a2; and a diagrammatic representation of the a2 VWFA domain sequence indicating the location of the mutations The percent binding for the chimera in which JA203 binding was abolished is given in bold, and the a3 helix in which it located is shaded The site of the E318W mutation is shown with an asterisk Binding to human wild-type a2, 100 ± 1.9%; binding to mouse wild-type a2, )0.8 ± 0.3% Microtitre plates were coated with 5 lgÆmL)1wild-type or chimeric VWFA domain and the binding of antibody JA203 measured Binding of anti-GST antiserum to constructs was used to normalize the data between VWFA domains Data are means ± SD, n ¼ 4 from two experiments.
Fig 7 a2-E318W shows elevated and specific binding to collagen IV (CB3 fragment), collagen I C-propeptide, laminin and E-cadherin (A) a2-E318W shows elevated binding to a2b1 ligands compared with wild-type VWFA domain Microtitre plates were coated with ligands and the binding of a2-E318W (grey bars, white bars) and wild-type (black bars, hatched bars) was measured in the presence of 1 m M
MgCl 2 (grey bars, black bars) or 1 m M MgCl 2 /10 m M EDTA (white bars, hatched bars) Data are means ± SD; n ‡ 6 from at least three experiments (B) Binding of a2-E318W to a2b1 ligands is specific Uninhibited binding (black bars); binding in the presence of the inhibitory peptide GFOGER (grey bars); binding in the presence of the control collagen peptide (white bars) Data are means ± SD; n ¼ 4 from two experiments Microtitre plates were coated with proteins (Collagen IV CB3 fragment, 3 lgÆmL)1; collagen I C-propeptide,
10 lgÆmL)1; laminin, 20 lgÆmL)1; E-cadherin-COMP, 10 lgÆmL)1;
50 kDa fragment of fibronectin, 10 lgÆmL)1; fibrinogen, 10 lgÆmL)1) and the binding of 0.5 lgÆmL)1fusion protein measured.
Trang 7the conformation of the cation coordinating residues seem
likely These data suggest that there is a general alteration in
the structure or environment of the ligand binding site as a
result of the E318W mutation, affecting the way in which
the tertiary complex of VWFA domain, cation and ligand
are formed This was accomplished without any alteration
in the specificity of the interaction The differential binding
of antibody JA203 to a2 E318W compared with wild-type
clearly identified this region as undergoing conformational
changes as a result of the mutation It is likely that this is due
to alterations at the MIDAS, as the C-terminal region of the
JA203 epitope falls within the ligand binding site These
changes in the MIDAS are likely to be quite subtle, as
antibodies JA202, JA208 and Gi9 map to this region [9], but
their binding was unchanged We previously showed that
the helix a3 region is very sensitive to events at the MIDAS,
as binding of JA208 is enhanced by collagen I, indicating an
interplay between the MIDAS and the JA208 epitope [9],
while JA202 and Gi9 inhibited ligand binding The JA203
data further demonstrate the importance of the helix a3
region in a2 VWFA domain function The E318W
muta-tion therefore has significant effects on VWFA domain
function, particularly on the MIDAS However, the fact
that E318 is spatially diatant from the MIDAS/JA203
epitope indicates that these effects do not occur through a
direct contribution of E318 to the MIDAS We therefore
propose that E318W functions by affecting the
conforma-tion of the VWFA domain This would be a highly specific
effect, since the global conformation of the domain is
unaffected, as shown by unaltered binding of a range of
antibodies to the mutant domain compared to the
wild-type
Residue E318 differs substantially in its position between
the open and closed forms of a2 A number of recent reports
have shown that the ligand binding function of the aL and
aM VWFA domains can be modulated by controlling the
conformational state of the domain The majority of
approaches have directly targeted the a7 helix, because of
the large difference in conformation between the two forms,
with the aim of stabilizing the open or closed forms
[35,36,42–44] Our report indicates that the general
approach of targeting a residue which undergoes a
confor-mational change between the open and closed forms can
also result in a gain of function in a2 This is the first report
of a deliberate engineering strategy being used for a2 and
since the E318 is conserved in a1, a10 and a11, the
gain-of-function property seen here should be reproducible in these
other integrins
To account for the molecular events resulting from the
F302W mutation [36], it was suggested that the increased
bulk of the tryptophan side chain drove residue 302 from its
buried location in the closed form, to the solvent-exposed
location seen in the open form, thus promoting the open
form of the whole domain [36] Residue E318 in a2 VWFA
domain is, like aM F302, buried in the closed form and
exposed in the open form, but our data indicate that the side
chain bulk is not important in the gain-of-function effect
However, the side chain of E318 forms a hydrogen bond
with R288 in the closed form, and this bond is broken on
moving to the open form The loss of the hydrogen bond in
the E318I/Y/W mutations, coupled with the hydrophobic
character of the mutation, may facilitate conformational
changes in the domain, for example by lowering the energy
barrier separating the two forms and thus promoting the open state
a2-E318 VWFA domain showed increased binding to collagen I C-propeptide, laminin and E-cadherin, compared with wild-type a2, indicating that the mutation also affected binding to these noncollagenous ligands The interactions with laminin and E-cadherin are normally very weak and in consequence hard to study However, the elevated binding seen with the mutant made possible inhibition studies and
we could show that the interaction of the a2 VWFA domain with these proteins could be inhibited by the GFOGER collagen peptide The residues responsible for the interac-tion of these proteins with the a2 VWFA domain have yet
to be identified, but our data suggest that glutamate residues are good candidates for the key cation-coordinating residues E31 of E-cadherin is known to be responsible for binding to aEb7 [45] and so this residue may also be central
to the interaction of E-cadherin with the a2 VWFA domain The availability of the a2-E318W mutant will greatly facilitate the mapping of the integrin binding sites on collagen C-propeptide, laminin and E-cadherin and will also
be a useful tool for the development of potent a2b1 antagonists
In conclusion, we have generated a mutant form of the a2 VWFA domain which shows a gain-of-function, and which may result in the promotion of the open form Helix a7 is therefore seen to be a valid target for such mutations across the integrin family This mutation will be of considerable value for future studies of the role of a2b1 at both the molecular and cellular level
A C K N O W L E D G E M E N T S
D T is supported by a Biotechnology and Biological Sciences Research Council Advanced Research Fellowship (34/AF09035);
M J H is a Wellcome Trust Principal Fellow; R W F is supported
by the Medical Research Council The authors are grateful to
M Brannan, S Craig and L Smith for advice and assistance.
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