Identification of the Collagen-binding Site of the von Willebrand Factor A3-domain
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m006548200
ISSN1083-351X
AutoresRoland A. Romijn, Barend Bouma, Winnifred Wuyster, Piet Gros, Jan Kroon, Jan J. Sixma, Eric G. Huizinga,
Tópico(s)Blood groups and transfusion
ResumoVon Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962–966, 981–997, and 1022–1026. Alanine mutants were constructed of residues Arg963, Glu987, His990, Arg1016, and His1023, located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His1023 abolished collagen binding, whereas mutation of Arg963 and Arg1016 reduced collagen binding by 25–35%. These residues are part of loops α3β4 and α1β2 and α-helix 3, respectively, and lie near the bottom face of the domain. His1023 and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains.AF2865871FE8 Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962–966, 981–997, and 1022–1026. Alanine mutants were constructed of residues Arg963, Glu987, His990, Arg1016, and His1023, located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His1023 abolished collagen binding, whereas mutation of Arg963 and Arg1016 reduced collagen binding by 25–35%. These residues are part of loops α3β4 and α1β2 and α-helix 3, respectively, and lie near the bottom face of the domain. His1023 and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains. AF2865871FE8 von Willebrand factor cystine knot phosphate-buffered saline bovine serum albumin asymmetric unit complementary determining region enzyme linked immunosorbent assay baby hamster kidney cells overexpressing furin metal ion dependent adhesion site noncrystallographic symmetry selenomethionine wild-type Platelet adhesion to damaged vessel walls is the first step in the formation of an occluding platelet plug, which leads to the arrest of bleeding during normal hemostasis. Platelet adhesion can also cause thrombotic complications such as the occlusion of atherosclerotic arteries (1Vischer U.M. De Moerloose P. Crit. Rev. Oncol. Hematol. 1999; 30: 93-109Crossref PubMed Scopus (33) Google Scholar). The multimeric glycoprotein von Willebrand factor (vWF)1 plays an essential role in platelet adhesion under conditions of high shear stress (2Ruggeri Z.M. Ware J. FASEB. J. 1993; 7: 308-316Crossref PubMed Scopus (268) Google Scholar, 3Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1115) Google Scholar). In this process vWF serves as a molecular bridge that links collagen exposed by the damaged vessel wall to glycoprotein Ib located on the platelet surface. Collagens that act as binding sites for vWF include types I and III in perivascular connective tissue and type VI in the subendothelial matrix (3Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1115) Google Scholar, 4Ruggeri Z.M. Thromb. Haemost. 1999; 82: 576-584Crossref PubMed Scopus (175) Google Scholar).Mature vWF consists of a 2050-residue monomer that contains multiple copies of so-called A, B, C, and D type domains and one CK (cystine knot) domain arranged in the order D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (1Vischer U.M. De Moerloose P. Crit. Rev. Oncol. Hematol. 1999; 30: 93-109Crossref PubMed Scopus (33) Google Scholar, 3Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1115) Google Scholar). Disulfide bond formation between N-terminal D3 domains and between C-terminal CK domains generates vWF multimers that consist of up to 80 monomers. The A1 domain contains the binding site for glycoprotein Ib (5Mohri H. Yoshioka A. Zimmerman T.S. Ruggeri Z.M. J. Biol. Chem. 1989; 264: 17361-17367Abstract Full Text PDF PubMed Google Scholar). The A3 domain (residues 920–1111) contains the major binding site for collagen types I and III (6Lankhof H. van Hoeij M. Schiphorst M.E. Bracke M. Wu Y.P. IJsseldijk M.J. Vink T. de Groot P.G. Sixma J.J. Thromb. Haemost. 1996; 75: 950-958Crossref PubMed Scopus (130) Google Scholar). The multimeric structure of vWF is essential for high affinity collagen binding (7Fischer B.E. Kramer G. Mitterer A. Grillberger L. Reiter M. Mundt W. Dorner F. Eibl J. Thromb. Res. 1996; 84: 55-66Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Multimeric vWF binds collagen with an apparent Kd of 1–7 nm (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar), while a recombinant A3 domain has a much higher Kd of 2 μm (9Cruz M.A. Yuan H. Lee J.R. Wise R.J. Handin R.I. J. Biol. Chem. 1995; 270: 10822-10827Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Deletion of the A2 and D4 domains, which flank the A3 domain, or deletion of the A1 domain do not decrease collagen binding of multimeric vWF (6Lankhof H. van Hoeij M. Schiphorst M.E. Bracke M. Wu Y.P. IJsseldijk M.J. Vink T. de Groot P.G. Sixma J.J. Thromb. Haemost. 1996; 75: 950-958Crossref PubMed Scopus (130) Google Scholar, 8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar). These data show that a monomeric A3 domain contains a fully active collagen-binding site, the only requirement for tight binding to collagen being the presence of multiple A3 domains within one vWF multimer.Integrin I-type domains are homologous to vWF A-type domains (10Colombatti A. Bonaldo P. Blood. 1991; 77: 2305-2315Crossref PubMed Google Scholar, 11Perkins S.J. Smith K.F. Williams S.C. Haris P.I. Chapman D. Sim R.B. J. Biol. Chem. 1994; 238: 104-119Google Scholar). I domains of integrin α-chains α1, α2, α10, and α11 all possess collagen-binding sites. A crystal structure of the α2-I domain reveals binding of a collagen-like peptide to a groove in the surface of the “top” face of the domain (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (824) Google Scholar). This groove contains a so-called metal ion-dependent adhesion site (MIDAS) (13Lee J.O. Rieu P. Arnaout M.A. Liddington R.C. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar,14Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (316) Google Scholar), which engages a glutamate residue of collagen.The location of the collagen-binding site in the vWF-A3 domain is not known. Crystal structures of A3 do not display a collagen-binding groove in the top face, instead, the surface of A3 is rather smooth (15Huizinga E.G. Van der Plas R.M. Kroon J. Sixma J.J. Gros P. Structure. 1997; 5: 1147-1156Abstract Full Text Full Text PDF PubMed Google Scholar, 16Bienkowska J. Cruz M.A. Atiemo A. Handin R.I. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Although the MIDAS motif is partly conserved, binding of A3 to collagen does not require a metal ion (17Pietu G. Fressinaud E. Girma J.P. Nieuwenhuis H.K. Rothschild C. Meyer D. J. Lab. Clin. Med. 1987; 109: 637-646PubMed Google Scholar, 18Bockenstedt P.L. McDonagh J. Handin R.I. J. Clin. Invest. 1986; 78: 551-556Crossref PubMed Scopus (43) Google Scholar), and no metal ion is observed in crystal structures of A3. Moreover, point mutations in the MIDAS motif of A3 do not disrupt collagen binding (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar, 16Bienkowska J. Cruz M.A. Atiemo A. Handin R.I. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) showing that the motif is not involved in collagen binding, at all. Site-directed mutagenesis studies of other residues in the top face of A3 have yielded conflicting results. Cruz et al. (19Cruz M.A. Bienkowska J. Mato A. Liddington R. Handin R.I. Blood Supplement I. 1997; 90 (.a Abstract): 23Google Scholar) reported in abstract form that amino acid substitutions D1069R, R1074D, R1090D, and E1092R resulted in a 50% reduction in binding of monomeric A3 to collagen. Van der Plas et al. (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar), however, observed normal collagen binding of fully processed multimeric vWF containing mutations D1069R, D1069A, or R1074A. In the same study, mutations V1040A/V1042A, D1046A, and D1066A also displayed normal collagen binding, suggesting that the collagen-binding site of vWF-A3 is not located in its top face.The crystallographic study presented here was conducted to provide new clues on the location of the collagen-binding site of the vWF-A3 domain. We determined the structure of the A3 domain in complex with a Fab fragment of monoclonal antibody RU5, which inhibits binding of vWF to collagen. Site-directed mutagenesis of residues located in the epitope region show that the collagen-binding site is located distant from the top face of A3.DISCUSSIONThe current study was aimed at locating the collagen-binding site of the vWF-A3 domain. For this purpose we solved the crystal structure of A3 in complex with a Fab fragment of RU5 that inhibits collagen binding. The structure of the complex shows that RU5 binds to residues within A3 sequences 962–966, 981–997, and 1022–1026. These residues are located in α-helix 2 and in loops α1β2, β3α2, and α3β4 at the bottom of one of the side faces of the A3 domain (see Fig. 3). Comparison of structures of A3 shows that RU5 binding does not induce long range conformational changes. This excludes a mechanism in which RU5-induced conformational changes inhibit collagen binding. It seems likely, therefore, that RU5 inhibits collagen binding by steric hindrance, which implies that the collagen-binding site is located at or close to the RU5 epitope.To confirm the location of the collagen-binding site, we constructed five charged-to-alanine mutations of residues located in or close to the RU5 epitope. The multimer distribution of these mutants was similar to that of wild-type vWF. Therefore, observed differences in collagen binding are not caused by the known dependence of collagen binding on vWF multimer size (7Fischer B.E. Kramer G. Mitterer A. Grillberger L. Reiter M. Mundt W. Dorner F. Eibl J. Thromb. Res. 1996; 84: 55-66Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). All five mutants bound normally to RU5, which shows that none of the mutated residues plays a dominant role in the A3·RU5 interaction and, more importantly, that the conformation of A3 in the neighborhood of the epitope and the collagen-binding site is not disturbed.Mutation H1023A abolished collagen binding almost completely, residual binding being similar to that observed for ΔA3-vWF, a deletion mutant that lacks the entire A3 domain. Therefore, His1023 plays a central role in A3-mediated collagen binding. His1023 is located in loop α3β4 and lies at the edge between the “front” face of the domain, formed by helices α2 and α3 and strand β3, and the bottom face, which is composed of several loops and contains the N and C termini (Fig. 7). A small reduction of collagen binding was observed for mutants R963A and R1016A located in the bottom and front face of the domain, respectively.Interestingly, His1023 and flanking residues display a large variety of conformations among eight models of A3 (Figs. 4 and7). In some A3 structures His1023 protrudes prominently from the surface of the domain, which may be a favorable position for interaction with collagen. Multiple conformations are also observed for loop β3α2. Like His1023, loop β3α2 is located at the edge between the front and bottom faces of A3. Because we did not mutate residues in loop β3α2, its involvement in collagen binding remains to be established. The observed flexibility of His1023 suggests that collagen binding may involve an induced-fit mechanism in which significant conformational changes occur in loop α3β4 upon binding of A3 to collagen.The amino acid sequence of collagen that is recognized by vWF-A3 has not yet been identified. We hypothesized previously that negatively charged residues in A3 could interact with basic residues on collagen (15Huizinga E.G. Van der Plas R.M. Kroon J. Sixma J.J. Gros P. Structure. 1997; 5: 1147-1156Abstract Full Text Full Text PDF PubMed Google Scholar). Residues now implicated in collagen binding are positively charged. Therefore, interaction of A3 with negatively charged residues on collagen appears more likely.In contrast to binding sites of other collagen binding domains, like the α1 and α2-I domains and the A domain ofStaphylococcus aureus adhesin (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (824) Google Scholar, 38Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Yang V.W.C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), the collagen-binding region of A3 does not have a groove or trench that could accommodate a collagen triple helix. The front face of the domain, harboring Arg1016, is rather flat. The bottom face, which contains Arg963 is less smooth, but no groove is present. Docking of a collagen triple helix on the A3 domain is not straightforward. In particular, it is not obvious how His1023, Arg963, and Arg1016 could simultaneously contact a triple helix in an extended conformation. To define the collagen-binding site more precisely, characterization of additional mutants will be necessary.Previously, the collagen-binding site of A3 was proposed to be located at its top face (15Huizinga E.G. Van der Plas R.M. Kroon J. Sixma J.J. Gros P. Structure. 1997; 5: 1147-1156Abstract Full Text Full Text PDF PubMed Google Scholar, 19Cruz M.A. Bienkowska J. Mato A. Liddington R. Handin R.I. Blood Supplement I. 1997; 90 (.a Abstract): 23Google Scholar) similar to the homologous I domains of integrins α1β1 and α2β1 (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (824) Google Scholar, 39Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 40Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 41Smith C. Estavillo D. Emsley J. Bankston L.A. Liddington R.C. Cruz M.A. J. Biol. Chem. 2000; 275: 4205-4209Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Point mutations introduced in the top face of an A3 monomer (19Cruz M.A. Bienkowska J. Mato A. Liddington R. Handin R.I. Blood Supplement I. 1997; 90 (.a Abstract): 23Google Scholar) and in multimeric vWF (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar) gave conflicting results. Our results now show conclusively that the collagen-binding site is located close to the bottom face and not in the top face of A3.Although our results rule out a role for the top face of the A3 domain in collagen binding, this side of the molecule may still be engaged in other interactions, such as binding of the A1 domain. Interaction between A1 and A3 has been suggested to play a role in activation of the A1 domain for binding to platelet receptor glycoprotein Ib (42Obert B. Houllier A. Meyer D. Girma J.P. Blood. 1999; 93: 1959-1968Crossref PubMed Google Scholar). Interesting in this respect are the buried and solvent-exposed conformation observed for residue Phe939, which is located close to the vestigial MIDAS motif in the top face of the domain (Fig.4 B). Solvent exposure of Phe939 has been proposed to stabilize the buried Asp934 of the vestigial MIDAS motif in the absence of a bound metal ion (16Bienkowska J. Cruz M.A. Atiemo A. Handin R.I. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Our observation of a buried conformation shows that exposure of Phe939 is not critical for structural stability. The two conformations of Phe939 may, however, be relevant for the putative interaction between A1 and A3, because the shape and hydrophobicity of the upper surface of A3 differs significantly between the solvent-exposed and buried conformation.In conclusion, the collagen-binding site of vWF-A3 is distinctly different from collagen-binding sites of I domains of integrins α1β1 and α2β1. vWF-A3 residues involved in collagen binding are located close to the bottom face of the domain. His1023 is essential for collagen binding, whereas Arg963 and Arg1016play ancillary roles. Multiple conformations observed for His1023 and adjacent residues suggest that binding of A3 to collagen involves an induced-fit mechanism. Platelet adhesion to damaged vessel walls is the first step in the formation of an occluding platelet plug, which leads to the arrest of bleeding during normal hemostasis. Platelet adhesion can also cause thrombotic complications such as the occlusion of atherosclerotic arteries (1Vischer U.M. De Moerloose P. Crit. Rev. Oncol. Hematol. 1999; 30: 93-109Crossref PubMed Scopus (33) Google Scholar). The multimeric glycoprotein von Willebrand factor (vWF)1 plays an essential role in platelet adhesion under conditions of high shear stress (2Ruggeri Z.M. Ware J. FASEB. J. 1993; 7: 308-316Crossref PubMed Scopus (268) Google Scholar, 3Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1115) Google Scholar). In this process vWF serves as a molecular bridge that links collagen exposed by the damaged vessel wall to glycoprotein Ib located on the platelet surface. Collagens that act as binding sites for vWF include types I and III in perivascular connective tissue and type VI in the subendothelial matrix (3Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1115) Google Scholar, 4Ruggeri Z.M. Thromb. Haemost. 1999; 82: 576-584Crossref PubMed Scopus (175) Google Scholar). Mature vWF consists of a 2050-residue monomer that contains multiple copies of so-called A, B, C, and D type domains and one CK (cystine knot) domain arranged in the order D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (1Vischer U.M. De Moerloose P. Crit. Rev. Oncol. Hematol. 1999; 30: 93-109Crossref PubMed Scopus (33) Google Scholar, 3Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1115) Google Scholar). Disulfide bond formation between N-terminal D3 domains and between C-terminal CK domains generates vWF multimers that consist of up to 80 monomers. The A1 domain contains the binding site for glycoprotein Ib (5Mohri H. Yoshioka A. Zimmerman T.S. Ruggeri Z.M. J. Biol. Chem. 1989; 264: 17361-17367Abstract Full Text PDF PubMed Google Scholar). The A3 domain (residues 920–1111) contains the major binding site for collagen types I and III (6Lankhof H. van Hoeij M. Schiphorst M.E. Bracke M. Wu Y.P. IJsseldijk M.J. Vink T. de Groot P.G. Sixma J.J. Thromb. Haemost. 1996; 75: 950-958Crossref PubMed Scopus (130) Google Scholar). The multimeric structure of vWF is essential for high affinity collagen binding (7Fischer B.E. Kramer G. Mitterer A. Grillberger L. Reiter M. Mundt W. Dorner F. Eibl J. Thromb. Res. 1996; 84: 55-66Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Multimeric vWF binds collagen with an apparent Kd of 1–7 nm (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar), while a recombinant A3 domain has a much higher Kd of 2 μm (9Cruz M.A. Yuan H. Lee J.R. Wise R.J. Handin R.I. J. Biol. Chem. 1995; 270: 10822-10827Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Deletion of the A2 and D4 domains, which flank the A3 domain, or deletion of the A1 domain do not decrease collagen binding of multimeric vWF (6Lankhof H. van Hoeij M. Schiphorst M.E. Bracke M. Wu Y.P. IJsseldijk M.J. Vink T. de Groot P.G. Sixma J.J. Thromb. Haemost. 1996; 75: 950-958Crossref PubMed Scopus (130) Google Scholar, 8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar). These data show that a monomeric A3 domain contains a fully active collagen-binding site, the only requirement for tight binding to collagen being the presence of multiple A3 domains within one vWF multimer. Integrin I-type domains are homologous to vWF A-type domains (10Colombatti A. Bonaldo P. Blood. 1991; 77: 2305-2315Crossref PubMed Google Scholar, 11Perkins S.J. Smith K.F. Williams S.C. Haris P.I. Chapman D. Sim R.B. J. Biol. Chem. 1994; 238: 104-119Google Scholar). I domains of integrin α-chains α1, α2, α10, and α11 all possess collagen-binding sites. A crystal structure of the α2-I domain reveals binding of a collagen-like peptide to a groove in the surface of the “top” face of the domain (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (824) Google Scholar). This groove contains a so-called metal ion-dependent adhesion site (MIDAS) (13Lee J.O. Rieu P. Arnaout M.A. Liddington R.C. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar,14Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (316) Google Scholar), which engages a glutamate residue of collagen. The location of the collagen-binding site in the vWF-A3 domain is not known. Crystal structures of A3 do not display a collagen-binding groove in the top face, instead, the surface of A3 is rather smooth (15Huizinga E.G. Van der Plas R.M. Kroon J. Sixma J.J. Gros P. Structure. 1997; 5: 1147-1156Abstract Full Text Full Text PDF PubMed Google Scholar, 16Bienkowska J. Cruz M.A. Atiemo A. Handin R.I. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Although the MIDAS motif is partly conserved, binding of A3 to collagen does not require a metal ion (17Pietu G. Fressinaud E. Girma J.P. Nieuwenhuis H.K. Rothschild C. Meyer D. J. Lab. Clin. Med. 1987; 109: 637-646PubMed Google Scholar, 18Bockenstedt P.L. McDonagh J. Handin R.I. J. Clin. Invest. 1986; 78: 551-556Crossref PubMed Scopus (43) Google Scholar), and no metal ion is observed in crystal structures of A3. Moreover, point mutations in the MIDAS motif of A3 do not disrupt collagen binding (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar, 16Bienkowska J. Cruz M.A. Atiemo A. Handin R.I. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) showing that the motif is not involved in collagen binding, at all. Site-directed mutagenesis studies of other residues in the top face of A3 have yielded conflicting results. Cruz et al. (19Cruz M.A. Bienkowska J. Mato A. Liddington R. Handin R.I. Blood Supplement I. 1997; 90 (.a Abstract): 23Google Scholar) reported in abstract form that amino acid substitutions D1069R, R1074D, R1090D, and E1092R resulted in a 50% reduction in binding of monomeric A3 to collagen. Van der Plas et al. (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar), however, observed normal collagen binding of fully processed multimeric vWF containing mutations D1069R, D1069A, or R1074A. In the same study, mutations V1040A/V1042A, D1046A, and D1066A also displayed normal collagen binding, suggesting that the collagen-binding site of vWF-A3 is not located in its top face. The crystallographic study presented here was conducted to provide new clues on the location of the collagen-binding site of the vWF-A3 domain. We determined the structure of the A3 domain in complex with a Fab fragment of monoclonal antibody RU5, which inhibits binding of vWF to collagen. Site-directed mutagenesis of residues located in the epitope region show that the collagen-binding site is located distant from the top face of A3. DISCUSSIONThe current study was aimed at locating the collagen-binding site of the vWF-A3 domain. For this purpose we solved the crystal structure of A3 in complex with a Fab fragment of RU5 that inhibits collagen binding. The structure of the complex shows that RU5 binds to residues within A3 sequences 962–966, 981–997, and 1022–1026. These residues are located in α-helix 2 and in loops α1β2, β3α2, and α3β4 at the bottom of one of the side faces of the A3 domain (see Fig. 3). Comparison of structures of A3 shows that RU5 binding does not induce long range conformational changes. This excludes a mechanism in which RU5-induced conformational changes inhibit collagen binding. It seems likely, therefore, that RU5 inhibits collagen binding by steric hindrance, which implies that the collagen-binding site is located at or close to the RU5 epitope.To confirm the location of the collagen-binding site, we constructed five charged-to-alanine mutations of residues located in or close to the RU5 epitope. The multimer distribution of these mutants was similar to that of wild-type vWF. Therefore, observed differences in collagen binding are not caused by the known dependence of collagen binding on vWF multimer size (7Fischer B.E. Kramer G. Mitterer A. Grillberger L. Reiter M. Mundt W. Dorner F. Eibl J. Thromb. Res. 1996; 84: 55-66Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). All five mutants bound normally to RU5, which shows that none of the mutated residues plays a dominant role in the A3·RU5 interaction and, more importantly, that the conformation of A3 in the neighborhood of the epitope and the collagen-binding site is not disturbed.Mutation H1023A abolished collagen binding almost completely, residual binding being similar to that observed for ΔA3-vWF, a deletion mutant that lacks the entire A3 domain. Therefore, His1023 plays a central role in A3-mediated collagen binding. His1023 is located in loop α3β4 and lies at the edge between the “front” face of the domain, formed by helices α2 and α3 and strand β3, and the bottom face, which is composed of several loops and contains the N and C termini (Fig. 7). A small reduction of collagen binding was observed for mutants R963A and R1016A located in the bottom and front face of the domain, respectively.Interestingly, His1023 and flanking residues display a large variety of conformations among eight models of A3 (Figs. 4 and7). In some A3 structures His1023 protrudes prominently from the surface of the domain, which may be a favorable position for interaction with collagen. Multiple conformations are also observed for loop β3α2. Like His1023, loop β3α2 is located at the edge between the front and bottom faces of A3. Because we did not mutate residues in loop β3α2, its involvement in collagen binding remains to be established. The observed flexibility of His1023 suggests that collagen binding may involve an induced-fit mechanism in which significant conformational changes occur in loop α3β4 upon binding of A3 to collagen.The amino acid sequence of collagen that is recognized by vWF-A3 has not yet been identified. We hypothesized previously that negatively charged residues in A3 could interact with basic residues on collagen (15Huizinga E.G. Van der Plas R.M. Kroon J. Sixma J.J. Gros P. Structure. 1997; 5: 1147-1156Abstract Full Text Full Text PDF PubMed Google Scholar). Residues now implicated in collagen binding are positively charged. Therefore, interaction of A3 with negatively charged residues on collagen appears more likely.In contrast to binding sites of other collagen binding domains, like the α1 and α2-I domains and the A domain ofStaphylococcus aureus adhesin (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (824) Google Scholar, 38Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Yang V.W.C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), the collagen-binding region of A3 does not have a groove or trench that could accommodate a collagen triple helix. The front face of the domain, harboring Arg1016, is rather flat. The bottom face, which contains Arg963 is less smooth, but no groove is present. Docking of a collagen triple helix on the A3 domain is not straightforward. In particular, it is not obvious how His1023, Arg963, and Arg1016 could simultaneously contact a triple helix in an extended conformation. To define the collagen-binding site more precisely, characterization of additional mutants will be necessary.Previously, the collagen-binding site of A3 was proposed to be located at its top face (15Huizinga E.G. Van der Plas R.M. Kroon J. Sixma J.J. Gros P. Structure. 1997; 5: 1147-1156Abstract Full Text Full Text PDF PubMed Google Scholar, 19Cruz M.A. Bienkowska J. Mato A. Liddington R. Handin R.I. Blood Supplement I. 1997; 90 (.a Abstract): 23Google Scholar) similar to the homologous I domains of integrins α1β1 and α2β1 (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (824) Google Scholar, 39Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 40Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 41Smith C. Estavillo D. Emsley J. Bankston L.A. Liddington R.C. Cruz M.A. J. Biol. Chem. 2000; 275: 4205-4209Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Point mutations introduced in the top face of an A3 monomer (19Cruz M.A. Bienkowska J. Mato A. Liddington R. Handin R.I. Blood Supplement I. 1997; 90 (.a Abstract): 23Google Scholar) and in multimeric vWF (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar) gave conflicting results. Our results now show conclusively that the collagen-binding site is located close to the bottom face and not in the top face of A3.Although our results rule out a role for the top face of the A3 domain in collagen binding, this side of the molecule may still be engaged in other interactions, such as binding of the A1 domain. Interaction between A1 and A3 has been suggested to play a role in activation of the A1 domain for binding to platelet receptor glycoprotein Ib (42Obert B. Houllier A. Meyer D. Girma J.P. Blood. 1999; 93: 1959-1968Crossref PubMed Google Scholar). Interesting in this respect are the buried and solvent-exposed conformation observed for residue Phe939, which is located close to the vestigial MIDAS motif in the top face of the domain (Fig.4 B). Solvent exposure of Phe939 has been proposed to stabilize the buried Asp934 of the vestigial MIDAS motif in the absence of a bound metal ion (16Bienkowska J. Cruz M.A. Atiemo A. Handin R.I. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Our observation of a buried conformation shows that exposure of Phe939 is not critical for structural stability. The two conformations of Phe939 may, however, be relevant for the putative interaction between A1 and A3, because the shape and hydrophobicity of the upper surface of A3 differs significantly between the solvent-exposed and buried conformation.In conclusion, the collagen-binding site of vWF-A3 is distinctly different from collagen-binding sites of I domains of integrins α1β1 and α2β1. vWF-A3 residues involved in collagen binding are located close to the bottom face of the domain. His1023 is essential for collagen binding, whereas Arg963 and Arg1016play ancillary roles. Multiple conformations observed for His1023 and adjacent residues suggest that binding of A3 to collagen involves an induced-fit mechanism. The current study was aimed at locating the collagen-binding site of the vWF-A3 domain. For this purpose we solved the crystal structure of A3 in complex with a Fab fragment of RU5 that inhibits collagen binding. The structure of the complex shows that RU5 binds to residues within A3 sequences 962–966, 981–997, and 1022–1026. These residues are located in α-helix 2 and in loops α1β2, β3α2, and α3β4 at the bottom of one of the side faces of the A3 domain (see Fig. 3). Comparison of structures of A3 shows that RU5 binding does not induce long range conformational changes. This excludes a mechanism in which RU5-induced conformational changes inhibit collagen binding. It seems likely, therefore, that RU5 inhibits collagen binding by steric hindrance, which implies that the collagen-binding site is located at or close to the RU5 epitope. To confirm the location of the collagen-binding site, we constructed five charged-to-alanine mutations of residues located in or close to the RU5 epitope. The multimer distribution of these mutants was similar to that of wild-type vWF. Therefore, observed differences in collagen binding are not caused by the known dependence of collagen binding on vWF multimer size (7Fischer B.E. Kramer G. Mitterer A. Grillberger L. Reiter M. Mundt W. Dorner F. Eibl J. Thromb. Res. 1996; 84: 55-66Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). All five mutants bound normally to RU5, which shows that none of the mutated residues plays a dominant role in the A3·RU5 interaction and, more importantly, that the conformation of A3 in the neighborhood of the epitope and the collagen-binding site is not disturbed. Mutation H1023A abolished collagen binding almost completely, residual binding being similar to that observed for ΔA3-vWF, a deletion mutant that lacks the entire A3 domain. Therefore, His1023 plays a central role in A3-mediated collagen binding. His1023 is located in loop α3β4 and lies at the edge between the “front” face of the domain, formed by helices α2 and α3 and strand β3, and the bottom face, which is composed of several loops and contains the N and C termini (Fig. 7). A small reduction of collagen binding was observed for mutants R963A and R1016A located in the bottom and front face of the domain, respectively. Interestingly, His1023 and flanking residues display a large variety of conformations among eight models of A3 (Figs. 4 and7). In some A3 structures His1023 protrudes prominently from the surface of the domain, which may be a favorable position for interaction with collagen. Multiple conformations are also observed for loop β3α2. Like His1023, loop β3α2 is located at the edge between the front and bottom faces of A3. Because we did not mutate residues in loop β3α2, its involvement in collagen binding remains to be established. The observed flexibility of His1023 suggests that collagen binding may involve an induced-fit mechanism in which significant conformational changes occur in loop α3β4 upon binding of A3 to collagen. The amino acid sequence of collagen that is recognized by vWF-A3 has not yet been identified. We hypothesized previously that negatively charged residues in A3 could interact with basic residues on collagen (15Huizinga E.G. Van der Plas R.M. Kroon J. Sixma J.J. Gros P. Structure. 1997; 5: 1147-1156Abstract Full Text Full Text PDF PubMed Google Scholar). Residues now implicated in collagen binding are positively charged. Therefore, interaction of A3 with negatively charged residues on collagen appears more likely. In contrast to binding sites of other collagen binding domains, like the α1 and α2-I domains and the A domain ofStaphylococcus aureus adhesin (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (824) Google Scholar, 38Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Yang V.W.C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), the collagen-binding region of A3 does not have a groove or trench that could accommodate a collagen triple helix. The front face of the domain, harboring Arg1016, is rather flat. The bottom face, which contains Arg963 is less smooth, but no groove is present. Docking of a collagen triple helix on the A3 domain is not straightforward. In particular, it is not obvious how His1023, Arg963, and Arg1016 could simultaneously contact a triple helix in an extended conformation. To define the collagen-binding site more precisely, characterization of additional mutants will be necessary. Previously, the collagen-binding site of A3 was proposed to be located at its top face (15Huizinga E.G. Van der Plas R.M. Kroon J. Sixma J.J. Gros P. Structure. 1997; 5: 1147-1156Abstract Full Text Full Text PDF PubMed Google Scholar, 19Cruz M.A. Bienkowska J. Mato A. Liddington R. Handin R.I. Blood Supplement I. 1997; 90 (.a Abstract): 23Google Scholar) similar to the homologous I domains of integrins α1β1 and α2β1 (12Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (824) Google Scholar, 39Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 40Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 41Smith C. Estavillo D. Emsley J. Bankston L.A. Liddington R.C. Cruz M.A. J. Biol. Chem. 2000; 275: 4205-4209Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Point mutations introduced in the top face of an A3 monomer (19Cruz M.A. Bienkowska J. Mato A. Liddington R. Handin R.I. Blood Supplement I. 1997; 90 (.a Abstract): 23Google Scholar) and in multimeric vWF (8Van der Plas R.M. Gomes L. Marquart J.A. Vink T. Meijers J.C.M. de Groot Ph. Sixma J.J. Huizinga E.G. Thromb. Haemost. 2000; 84: 1005-1011Crossref PubMed Scopus (35) Google Scholar) gave conflicting results. Our results now show conclusively that the collagen-binding site is located close to the bottom face and not in the top face of A3. Although our results rule out a role for the top face of the A3 domain in collagen binding, this side of the molecule may still be engaged in other interactions, such as binding of the A1 domain. Interaction between A1 and A3 has been suggested to play a role in activation of the A1 domain for binding to platelet receptor glycoprotein Ib (42Obert B. Houllier A. Meyer D. Girma J.P. Blood. 1999; 93: 1959-1968Crossref PubMed Google Scholar). Interesting in this respect are the buried and solvent-exposed conformation observed for residue Phe939, which is located close to the vestigial MIDAS motif in the top face of the domain (Fig.4 B). Solvent exposure of Phe939 has been proposed to stabilize the buried Asp934 of the vestigial MIDAS motif in the absence of a bound metal ion (16Bienkowska J. Cruz M.A. Atiemo A. Handin R.I. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Our observation of a buried conformation shows that exposure of Phe939 is not critical for structural stability. The two conformations of Phe939 may, however, be relevant for the putative interaction between A1 and A3, because the shape and hydrophobicity of the upper surface of A3 differs significantly between the solvent-exposed and buried conformation. In conclusion, the collagen-binding site of vWF-A3 is distinctly different from collagen-binding sites of I domains of integrins α1β1 and α2β1. vWF-A3 residues involved in collagen binding are located close to the bottom face of the domain. His1023 is essential for collagen binding, whereas Arg963 and Arg1016play ancillary roles. Multiple conformations observed for His1023 and adjacent residues suggest that binding of A3 to collagen involves an induced-fit mechanism. We thank the staff of the EMBL Outstation DESY (Hamburg, Germany) for assistance in x-ray data collection.
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