A novel gainoffunction mutation of the integrin 2 VWFA domain

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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.
Eur. J. Biochem. 269, 1136–1144 (2002) Ó FEBS 2002

A 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; 2The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 3Department 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 Willebrand 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 hydrophobicity 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.

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 predisposition 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 hypersensitivity 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 specificity, 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-iondependent 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 binding 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 conformational 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: [email protected] Abbreviations: GST, glutathione S-transferase; MIDAS, metal iondependent adhesion site; VWFA domain, von Willebrand factor type A-domain. (Received 17 September 2001, revised 26 November 2001, accepted 14 December 2001)

Keywords: adhesion; collagen; extracellular matrix; integrin.

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possibly 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.

MATERIALS AND METHODS General reagents Acid soluble rat tail type I collagen and EHS-laminin were obtained from Sigma-Aldrich. Collagen peptides were prepared as described in [30]. Collagen IV fragment CB3 was the kind gift of K. Ku¨hn, Max-Planck-Institute for Biochemistry, 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 GCTCTACTAGAAAAG-3¢. The resulting doublestranded 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 biotinylated 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 vWFAdomain fusion proteins diluted in 136.8 mM NaCl, 8.1 mM

Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 0.9 mM CaCl2, 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)1 BSA, 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, 0.05 M NaH2PO4, 0.1 M 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)1 BSA at double the final concentration). 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 following 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, University of Manchester, UK) 10 lgÆmL)1 in 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)1 Chelex 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)1 BSA, and 50 lL cation (in NaCl/Tris, 1 mgÆmL)1 BSA at double the final concentration) was added followed by 50 lL VWFA domain (also in NaCl/Tris, 1 mgÆmL)1 BSA at double the final concentration). All subsequent steps were as above with the exception that buffer A contained 1 mM MgCl2 and 1 mM MnCl2. Assays to measure antibody binding to recombinant VWFA domains were carried out after the method of

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Brookman et al. [40]. Immulon 4 microtitre plates were coated with 100 lL 5 lgÆmL)1 fusion 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.

RESULTS 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

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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 interaction 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)1 and 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 wildtype (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.

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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)1 VWFA domains and the binding of biotinylated collagen I measured. Binding of mutants in the presence 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.

The E318W mutation affects the MIDAS and helix a3

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 mM MgCl2 (m,j) or 1 mM MgCl2/10 mM EDTA (n,h). Data are means ± SD; n ¼ 4 from two experiments. (B) Microtitre plates were coated with 1 lgÆmL)1 collagen I and binding of a2-E318W or wild-type a2 (2 lgÆmL)1) measured in the presence of 1 mM MgCl2 (dark bars) or 1 mM MgCl2/10 mM EDTA (light bars). Data are means ± SD; n ¼ 7 from three experiments (wild-type) and n ¼ 5 from two experiments (a2-E318W). (C) Microtitre plates were coated with 1 lgÆmL)1 collagen I and the binding of 0.5 lgÆmL)1 a2-E318W measured in the presence of 1 mM MgCl2 with the addition of 100 lgÆmL)1 GFOGER collagen peptide (which carries the a2 binding site), or control collagen peptide (which does not carry the a2 binding site).

E318R 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 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 concentrations (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

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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)1 wild-type a2 (B) and the binding of 1 lgÆmL)1 biotinylated 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)1 in the presence of Mn2+ (d) or Mg2+ (m) and detected with anti-GST Ig. Data are means ± range (n ¼ 2) from representative experiments.

that 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-cadherin [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. 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)1 VWFA domain and the binding of antibodies measured over a range of concentrations. Data are means±SD; n ¼ 6 from two experiments.

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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)1 wild-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.

a2-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.

DISCUSSION 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-offunction effect as well as 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. 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 mM MgCl2 (grey bars, black bars) or 1 mM MgCl2/10 mM 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)1 fusion protein measured.

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the 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 mutation 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 conformation 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 wildtype. 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 conformational 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-offunction 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 interaction 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.

ACKNOWLEDGEMENTS 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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