A novel gain-of-function mutation of the integrin a2 VWFA domain Alexis Aquilina 1 , Michelle Korda 1 , Jeffrey M. Bergelson 2 , Martin J. Humphries 1 , Richard W. Farndale 3 and Danny Tuckwell 1 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 r eceptor 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 h as 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-E3 18W VWFA domain showed elevated and specific b inding to collagen I compared with t he 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 V WFA 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 i nhibited by collagen p eptide containing the GFOGER motif, i ndicating that these bound to the VWFA domain by a similar mechanism t o collagen I. These data indicate that residue E318 plays a novel and important role in mod ulating a2 VWFA domain–ligand b inding and may b e 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 m ediate cell adhesion in vitro to a range of collagens [2–4], but is al so a r eceptor for a number of noncollagenous molecules including laminins, collagen C-propeptides, E-cadherin, and certain viruses a nd snake toxins [5–10] (J. Whittard, A. P. Mould, A. Koch, O. Pertz, J. Engel, M. J. Humphries, unpublished data). Binding o f a2b1 to c ollagen induces collagen a nd collagenase gene expression [11,12] and the initiation of the p 38 MAPK signalling pathway [4]. a2b1 is also responsible for f orce generation by cells in collage n 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 a lthough a2b1 i s probably not the major collagen receptor on platelets [17], a genetic predis- position to increased levels of platelet a2b1maybearisk factor for stroke [18], myocardial i nfarction [19], and diabetic retinopathy [20] (although see [21,22]). a2b1has also been found to be involved in the r egulation 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 v on Willebrand Factor type A domain (VWFA domain, also known a s the A- or I-domain) of t he 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 c ollagen 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 ind icate that similar conforma- tional changes in the aMandaL 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 a n increase in the proportion of active integrin [ 36]. This indicates that F302 plays an important role in modulating ligand b inding, 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: d anny.tuckwell@man.ac.uk Abbreviations:GST,glutathioneS-transferase; MIDAS, metal ion- dependent adhesion site; VWFA domain, von Willebrand factor type A-domain. (Received 1 7 September 2001, revised 26 November 2001, accepted 14 December 2001) Eur. J. Biochem. 269, 1136–1144 (2002) Ó FEBS 2002 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 e xamined 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 c ollagen and EHS-laminin w ere obtained from S igma-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]; t he 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, Switze rland. 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] a nd the BIO-RAD Muta-Gene mutagenesis kit. Essentially, single-stranded uracil-containing DNA was generated by helper phage infection o f Escherichia coli strainCJ236andthenusedas 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 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 b inding assays to measure binding of biotiny- lated collagen to a2 vWFA-domain were carried out as follows: Immulon 4 microtitre plates (Dynex, B illinghurst, West Sussex, UK) were coated with 100 lL a2 vWFA- domain fusion proteins diluted in 136.8 m M NaCl, 8.1 m M Na 2 HPO 4 ,2.7m M KCl, 1.5 m M KH 2 PO 4 ,0.9m M CaCl 2 , 0.5 m M MgCl 2 , pH 7.4 (NaCl/P i + wherethe+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 lL50mgÆmL )1 BSA,in40 m M Tris/HCl, 150 m M NaCl, pH 7.4 (NaCl/Tris), for 2 h at room temperature. Wells were then washed three times with 200 lL N aCl/Tris, 1 mgÆmL )1 BSA, 1 m M MgCl 2 (bufferA),andtoeachwellwasthenadded50lL inhibitor (in buffer A at double the final concentration) followed by 50 lL biotinylated collagen I (also in buffer A at double t he 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 b y the addition of 2 m M 2¢2¢-azino-bis- (3-ethylbenzthiazoline-6-sulphonic acid), 0.03% (v/v) H 2 O 2 , 0.05 M NaH 2 PO 4 ,0.1 M sodium acetate, pH 5.0 (ABTS reagent). Absorbance was measured at 405 nm on a plate reader. For experiments measuring collagen b inding 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, 1mgÆmL )1 BSA, then 50 lLcation(inNaCl/Tris, 1mgÆmL )1 BSA at double the final concentration) was added followed by 50 lL b iotinylated c ollagen I (also i n NaCl/Tris, 1 mgÆmL )1 BSA at double the final concentra- tion). All subsequent steps were as above. For the measurement o f 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 c ollagen I or other ligands diluted in NaCl/P i + overnight at 4 °C. The follow- ing day, protein solutions were removed and wells blocked as above. We lls were then washed three times with buffer A, andtoeachwellwasadded50lL inhibitor (in buffer A at double t he 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 timeswith200lLbufferAand100lL sheep anti-GST antiserum ( the kind gift of V. A llan and S. Taylor, Univer- sity of Manchester, UK) 10 lgÆmL )1 in buffer A added f or 1 h at room temperature. Wells were then washed thre e 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 thre e 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 % 1gÆL )1 Chelex 100 overnight a nd the Chelex removed by filtration p rior to use in assays. The assay protocol was carried out as above up to the b locking s tage, then wells were washed three times with 200 lL N aCl/Tris, 1 mgÆmL )1 BSA, and 50 lLcation(in NaCl/Tris, 1 mgÆmL )1 BSA at double the final concentra- tion) was a dded followed b y 50 lL VWFA domain (also in NaCl/Tris, 1 mgÆmL )1 BSA at double the final concentra- tion). All subsequent steps were as above with the exception that buffer A contained 1 m M MgCl 2 and 1 m M MnCl 2 . Assays to measure antibody binding to recombinant VWFA domains w ere carried out after the method of Ó FEBS 2002 Integrin a2 gain-of-function mutation (Eur. J. Biochem. 269) 1137 Brookman et al. [40]. Immulon 4 microtitre plates were coated with 100 lL5lgÆmL )1 fusion protein in NaCl/P i + , overnight at 4 °C. Wells were the n washed twice with 200 lLNaCl/P i – (NaCl/P i + without Ca 2+ or Mg 2+ )and blocked with 100 lL 2% (w/v) fat-free milk powder, NaCl/ P i – ,for1hat4°C. Wells were then washed twice with 200 lL 0.1% (v/v) Tween 20, NaCl/P i – ,and100lL antibody diluted in 0.5% ( w/v) milk powder, 0.1% (v/v) Tween 20, NaCl/P i – , added for 2 h at 4 °C. Wells were then washed twice with 200 lL 0.1% (v/v) Tween 20, NaCl/P i – , 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) T ween 20, NaCl/P i – , added for 2 h at 4 °C. Wells were then washed three times with 200 lL 0.1% (v/v) Tween 20, NaCl/P i – ,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 showntoresultinagainoffunctioninboththeisolatedaM VWFA domain and in aMb2 [36]. Comparison of the a2 and aM sequences and structures indicated that the homologous residue in a2isE318(Fig.1A),aresiduein the a7 helix which, like aM F302, undergoes a large displacement on collagen binding and moves fro m a buried to an exposed position (Fig. 1B and C). a2E318was therefore mutated to tryptophan, the recombinant mutant VWFA domain gen erated, and t he a2 E318W VWFA domain tested in solid phase binding assays. a2-E318W VWFA domain showed enhanced binding to collagen I compared with w ild-type VWFA domain, both w hen binding of biotinylate d collagen to i mmobilized 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. 2 A 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 t o VWFA domains were a nalysed by curve fitting a nd double reciprocal plots (analyses were carried out on the results of four independent experiments): the apparent affinities for the wild-type and mutant V WFA 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. 2 A, 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 b inding site (MIDAS). This increase in binding was not due t o misfolding or d ifferences in stability of t he 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 do main (data not shown). To determine the molecular basis of the effect of the E318W mutation, E318 was mutated to Y, I a nd R and recombinant VWFA domains tested: a2-E318Y and a2-E318I behaved similarly to a2-E318 W, 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 b inding, but the size of the r esidue is less important. The decreased b inding of Fig. 1. E318 moves between the ‘open’ and ‘closed’ forms of a2 VWFA domain. (A) Alignment of C-terminal seq uence of a2andaMVWFA domains sh owing a2E318andaM F302 (boxed). Secondary structural elements are marked above the alignment; dots indicate residues identical between a2andaM. (B, C) Structure of the C-terminal region of a2 VWFA domain, in the presence (B) or absence (C) of collagen p eptide ligated. The arrow indicates t he position of the E318 side chain, which is altered by the conformational change. 1138 A. Aquilina et al. ( Eur. J. Biochem. 269) Ó FEBS 2002 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 t he course of a few days. The E318W mutation affects the MIDAS and helix a3 The interac tion of c ollagens with the a2 VWFA domain requires the MIDAS c ation and we have previously shown that either Mg 2+ or Mn 2+ 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 f or binding of biotinylated collagen i n t he presence of Mg 2+ but differed in their binding in the p resence of Mn 2+ ,withthea2-E318W VWFA binding curve shifted to the left compared w ith the wild-type, which displayed a complex profile ( Fig. 4A and B). Similar profiles for a2-E318W and wild-type w ere seen over a range of cation and fusion protein concentra- tions (data not shown). A ssays in which t he 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 t he formation of t he collagen– cation–VWFA domain complex at the MIDAS, l eading 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 s creened 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 bo und to the w ild-type and mutant domains at s imilar levels (data f or JA202 is shown f or 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 p lates were c oated with 10 lgÆmL )1 a2-E318W (n,m) or wild-type a2(h,j) and the binding of bio tinyated collagen I measured in the presence of 1 m M MgCl 2 (m,j)or1m M MgCl 2 /10 m M EDTA (n,h). Data are means ± SD; n ¼ 4fromtwo experiments. (B) Microtitre plates were coated with 1 lgÆmL )1 colla- gen I and binding of a2-E318W or wild-type a2(2lgÆmL )1 )measured in the p resence 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 t wo experiments (a2-E318W). (C) Microtitre plates were c oated with 1 lgÆmL )1 collagen I and the binding of 0.5 lgÆmL )1 a2-E318W measured in the presence of 1 m M MgCl 2 with the ad dition of 100 lgÆmL )1 GFOGER collagen peptide (which carries the a2 binding site), or co ntrol 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 mutatio ns E318W (r), E318I (m) E 318Y (d)andE318R(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 pres- ence of EDTA was the same as that seen for wild-type + EDTA. Data are means ± SD; n ¼ 6 from t hree experiments except for E318R where n ¼ 4 from two experiments. Ó FEBS 2002 Integrin a2 gain-of-function mutation (Eur. J. Biochem. 269) 1139 that JA203 was raised i n mouse against a human antigen, and will therefore bind t o 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 t herefore 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 t opologically distinct from residue E318 and there is a number of chimeras which alter amino acids located between E318 and he lix a3withno 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 d ata 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 m ay 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 recep tor not only for collage n I but also for collagen I V, collagen I C-propeptide, laminin a nd 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 i ncrease i n binding to the control p roteins, fibrinogen or the 50 k Da fragmen t of fibronectin was seen. This indicated t hat the enhancing effect of the E318W mutation was not confined to collagens alone. Little i s 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 )1 wild-type a2 (B) and the binding of 1 lgÆmL )1 biotinylated collagen I measured in the presence of Mn 2+ (d), Mg 2+ (m), or EDTA (e). (C, D) Micro titre plates were coated with collagen I (1 lgÆmL )1 )anda2-E318W (C) or wild-type a2(D)addedat1lgÆmL )1 in the presence of M n 2+ (d)orMg 2+ (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-E318 W (s) than to wild-type a2 VWFA domain (d); (B) B inding 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 bin ding of antibodies measured over a range of con- centrations. Data a re means±SD; n ¼ 6 from two experiments. 1140 A. Aquilina et al. ( Eur. J. Biochem. 269) Ó FEBS 2002 a2-E318W to collagen IV C B3, C-propeptide, laminin and E-cadherin was inh ibited 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 o f the a2 VWFA domain. I ntroduction 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 t hat the mutation exerted its effect by inducing conformational changes in the VWFA domain. We a lso describe f urther mutations of E318 which help to define the molecular basis of the gain-of- function effect as well as fu nctional studies showing that noncollagenous a2b1 ligands bind to the mutant VWFA domain at an elevated level relative to wild-type a nd by the same mech anism a s c ollagen 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 c ocrystal [32]. This interaction involves a discrete set of residues clustering round the cation binding site, as well as the cation itself. It is o f interest t hat the E318W mutation has such a large effect on the binding of collagenous and non collagenous 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 Mg 2+ and M n 2+ are n ot clear, change s 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 s equence indicating the location of the mutations. The percent b inding 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 t o normalize the data between VWFA domains. Data are means ± SD, n ¼ 4 from t wo 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 plat es were coated with ligands and the binding o f a2-E318W (grey bars, white b ars) and w ild-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 co llagen 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, 2 0 lgÆmL )1 ;E-cadherin-COMP,10lgÆmL )1 ; 50 kDa fragment of fibronectin, 10 lgÆmL )1 ; fibrinogen, 10 lgÆmL )1 ) and the binding of 0.5 lgÆmL )1 fusion protein measured. Ó FEBS 2002 Integrin a2 gain-of-function mutation (Eur. J. Biochem. 269) 1141 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 i n which the t ertiary complex of VWFA domain, c ation and ligand are formed. This was accomplished without any a lteration in the specificity of t he interaction. The differential binding of antibody JA203 to a2 E318W compared with wild-type clearly identified this region as un dergoing conformational changes as a result of the mutation. It is likely that this is due to alterations at the MIDAS, as t he 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 sen sitive 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 b inding. The JA203 data further d emonstrate t he importance o f the helix a3 region in a2 V WFA domain function. The E318W muta- tion therefore h as significant effects o n VWFA domain function, particularly o n t he MIDAS. However, the fact that E318 is spatially diatant from the MIDAS/JA203 epitope indicates that these effects do not occur t hrough a direct contribution of E318 to the MIDAS. We therefore propose t hat E318W functions by affecting the conforma- tion of the VWFA d omain. 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 t he wild- type. Residue E318 differs substantially in its position between the open and closed forms of a2. A number of recent reports have shown t hat the ligand binding function of the aLand aM VWFA domains can be modulated by controlling the conformational state o f the domain. The majo rity 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 b etween the open a nd closed forms can also result in a gain of function in a2. This is t he first report of a deliberate engineering strategy being u sed for a2and since t he 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 a2VWFA domain is, like aM F302, buried in the closed f orm 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 brok en 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 t he 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 an d 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 i nhibition studies and we could show that the interaction of the a2 VWFA domain with these p roteins could b e inhibited by the GFOGER collagen peptide. The residues responsible for the interac- tion of these proteins with th e a2 VWFA domain have yet to be identified, but our data su ggest that glutamate residues are good c andidates for the key cation-coordinating residues. E31 of E-cadherin i s 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. 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