Echovirus 1 Interaction with the Human Very Late Antigen-2 (Integrin α2β1) I Domain

Abstract

The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1. Binding sites for both collagen and echovirus 1 have been mapped to the I domain within the α2 subunit of the VLA-2 α2β1 heterodimer. Although murine VLA-2 interacts with collagen, it does not bind virus. We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199–201 and 212–216, independently involved in virus attachment. These residues are distinct from the metal ion-dependent adhesion site previously demonstrated to be essential for VLA-2 interactions with collagen. Mutations in three metal ion-dependent adhesion site residues that abolish adhesion to collagen had no effect on virus binding. These results confirm that different sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands. The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1. Binding sites for both collagen and echovirus 1 have been mapped to the I domain within the α2 subunit of the VLA-2 α2β1 heterodimer. Although murine VLA-2 interacts with collagen, it does not bind virus. We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199–201 and 212–216, independently involved in virus attachment. These residues are distinct from the metal ion-dependent adhesion site previously demonstrated to be essential for VLA-2 interactions with collagen. Mutations in three metal ion-dependent adhesion site residues that abolish adhesion to collagen had no effect on virus binding. These results confirm that different sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands. The integrin VLA-2 1The abbreviations used are: VLA, very late antigen; MIDAS, metal ion-dependent adhesion site; GST, glutathione S-transferase; CR3, complement receptor 3 (CD11b/CD18, αMβ2, or Mac-1); CHO, Chinese hamster ovary; mAbs, monoclonal antibodies. (CD49b/CD29) mediates cell interactions with the extracellular matrix proteins collagen and laminin (1Hemler M.E. Annu. Rev. Immunol. 1990; 8: 365-400Crossref PubMed Google Scholar). Human VLA-2 is also the receptor for the human pathogen echovirus 1 (2Bergelson J.M. Shepley M.P. Chan B.M.C. Hemler M.E. Finberg R.W. Science. 1992; 255: 1718-1720Crossref PubMed Scopus (248) Google Scholar). VLA-2 is a heterodimer composed of a 150-kDa α2 and a 130-kDa β1 subunit (1Hemler M.E. Annu. Rev. Immunol. 1990; 8: 365-400Crossref PubMed Google Scholar). Within the α2 subunit, the 200-amino acid I (“inserted”) domain has sequence similarity to the I or A domains of such proteins as von Willebrand factor, complement proteins, cartilage matrix protein, and certain other integrins (3Colombatti A. Bonaldo P. Blood. 1991; 77: 2305-2315Crossref PubMed Google Scholar). Binding sites for both collagen and echovirus 1 have been mapped within the I domain (amino acids 140–349) of the human α2 subunit (4Bergelson J.M. St. John N.F. Kawaguchi S. Pasqualini R. Berdichevsky F. Hemler M.E. Finberg R.W. Cell Adhesion and Commun. 1994; 2: 455-464Crossref PubMed Scopus (51) Google Scholar, 5Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar). Monoclonal antibodies that block VLA-2 interactions with collagen and/or echovirus recognize epitopes within the I domain, between amino acids 173 and 259, suggesting that sites required for interactions with both ligands reside within this portion of the molecule (5Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar). Both echovirus 1 (6King S.L. Cunningham J.A. Finberg R.W. Bergelson J.M. J. Virol. 1995; 69: 3237-3239Crossref PubMed Google Scholar) and collagen (7Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 8Tuckwell D. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 108: 1629-1637Crossref PubMed Google Scholar) bind to the isolated human VLA-2 I domain expressed in bacteria as a glutathione S-transferase (GST) fusion protein. Other results suggest that the binding sites for collagen and virus, while both contained within the I domain, are not identical. Although some monoclonal antibodies block VLA-2 interaction with both collagen and virus, others inhibit binding to one ligand or the other (5Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar). Furthermore, a variety of evidence indicates that interactions with collagen and echovirus 1 are regulated by different mechanisms. In contrast to collagen binding to VLA-2, echovirus 1 interactions do not discriminate between functional forms of the receptor, and are not enhanced by activating antibodies or phorbol esters (9Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar). In addition, whereas VLA-2 interaction with collagen is magnesium-dependent, echovirus 1 attachment occurs in the absence of divalent cations (9Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar). Mutation of any of three aspartate or threonine residues within the VLA-2 I domain (Asp151, Thr221, and Asp254) abolishes cell adhesion to collagen (5Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 7Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar). Mutations at similar positions within the I domain (called by some investigators the A domain) of the β2 integrin CR3 (CD11b/CD18) ablate binding to CR3 ligands and to divalent cations (10Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 11Kamata T. Wright R. Takada Y. J. Biol. Chem. 1995; 270: 12531-12535Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The crystal structure of the CR3 I domain shows that these residues serve to coordinate a magnesium ion, and form what has been termed the metal ion-dependent adhesion site (MIDAS) (12Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (806) Google Scholar); a similar metal-binding site is evident in the crystal structure of the leukocyte function-associated antigen-1 (CD11a/CD18) I domain (13Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (290) Google Scholar, 14Qu A. Leahy D.J. Structure. 1996; 4: 931-942Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). A MIDAS motif, consisting of a DXSXS sequence (whereX represents any amino acid) plus noncontiguous threonine and aspartate residues, is present in a number of other I or A domains (12Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (806) Google Scholar). Because VLA-2 interactions with echovirus 1 are relatively cation-independent, it is not clear whether the VLA-2 MIDAS is essential for virus binding. Although both human and murine VLA-2 bind collagen, only human VLA-2 mediates echovirus 1 attachment and infection (1Hemler M.E. Annu. Rev. Immunol. 1990; 8: 365-400Crossref PubMed Google Scholar, 2Bergelson J.M. Shepley M.P. Chan B.M.C. Hemler M.E. Finberg R.W. Science. 1992; 255: 1718-1720Crossref PubMed Scopus (248) Google Scholar, 15Edelman J.M. Chan B.M.C. Uniyal S. Onodera H. Wang D.Z. Damjanovich L. Latzer D.B. Finberg R. Bergelson J.M. Cell Adhesion and Commun. 1994; 2: 131-143Crossref PubMed Scopus (11) Google Scholar). Experiments with human-murine chimeras have shown that specific virus attachment to human VLA-2 depends on the human α2 subunit, and on the human I domain (4Bergelson J.M. St. John N.F. Kawaguchi S. Pasqualini R. Berdichevsky F. Hemler M.E. Finberg R.W. Cell Adhesion and Commun. 1994; 2: 455-464Crossref PubMed Scopus (51) Google Scholar, 16Bergelson J.M. St. John N. Kawaguchi S. Chan M. Stubdal H. Modlin J. Finberg R.W. J. Virol. 1993; 67: 6847-6852Crossref PubMed Google Scholar). We have now identified sites within the I domain, distinct from the MIDAS residues involved in collagen adhesion, responsible for specific echovirus 1 attachment to human VLA-2. A 650-base pair fragment encoding the I domain (amino acids 140–349) was amplified from a murine α2 cDNA clone (15Edelman J.M. Chan B.M.C. Uniyal S. Onodera H. Wang D.Z. Damjanovich L. Latzer D.B. Finberg R. Bergelson J.M. Cell Adhesion and Commun. 1994; 2: 131-143Crossref PubMed Scopus (11) Google Scholar), using polymerase chain reaction primers that introduced BamHI and EcoRI restriction sites, and inserted into the pGEX-KT GST expression vector (17Hakes D.J. Dixon J.E. Anal. Biochem. 1992; 202: 293-298Crossref PubMed Scopus (222) Google Scholar). Chimeric I domain DNA fragments were created using splice-overlap extension polymerase chain reaction techniques (4Bergelson J.M. St. John N.F. Kawaguchi S. Pasqualini R. Berdichevsky F. Hemler M.E. Finberg R.W. Cell Adhesion and Commun. 1994; 2: 455-464Crossref PubMed Scopus (51) Google Scholar) with a modification designed to amplify only products containing mutations introduced in the first reaction (18Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). The sequence of each subcloned polymerase chain reaction product was verified using an automated Applied Biosystems sequencer. One additional human-murine chimeric I domain, combining human amino acids 140–218 and murine amino acids 219–349, was prepared using aPpuMI restriction site present in both murine and human cDNAs. GST fusion proteins were produced in Escherichia coli and purified on glutathione-Sepharose essentially as described (6King S.L. Cunningham J.A. Finberg R.W. Bergelson J.M. J. Virol. 1995; 69: 3237-3239Crossref PubMed Google Scholar). The CR3 I domain-GST fusion protein was provided by P. Rieu and M. A. Arnaout (10Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (318) Google Scholar). The D151A, T221A, and D254A mutant I domain-GST fusion proteins have been described (7Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar). [35S]Methionine-labeled echovirus 1, prepared as described (2Bergelson J.M. Shepley M.P. Chan B.M.C. Hemler M.E. Finberg R.W. Science. 1992; 255: 1718-1720Crossref PubMed Scopus (248) Google Scholar), was added either to purified fusion proteins immobilized on glutathione-Sepharose beads (5,000 cpm, 2 μg of protein/aliquot) or to cell monolayers in 24-well plates (20,000 cpm/well). Samples were incubated for 1 h at room temperature with rocking. Beads or cell monolayers were washed to removed unbound virus and dissolved for liquid scintillation counting. Additional aliquots of fusion proteins immobilized on glutathione-Sepharose beads were boiled in Laemmli sample buffer and run on SDS-polyacrylamide electrophoresis gels to confirm equal protein loading (not shown). A chimeric murine α2 construct with human amino acids at positions 199–216 was prepared in the expression vector pFNeo (19Hahn W.C. Menzin E. Saito T. Germain R.N. Bierer B.E. Gene (Amst.). 1993; 127: 267-268Crossref PubMed Scopus (35) Google Scholar). CHO cells were transfected by electroporation (280 V, 960 microfarads in a Bio-Rad Gene Pulser), and selected first in media containing 0.5 mg/ml geneticin, and then for α2 expression by panning two times on collagen-coated plastic dishes as described (15Edelman J.M. Chan B.M.C. Uniyal S. Onodera H. Wang D.Z. Damjanovich L. Latzer D.B. Finberg R. Bergelson J.M. Cell Adhesion and Commun. 1994; 2: 131-143Crossref PubMed Scopus (11) Google Scholar). CHO cells transfected with murine α2 and selected by panning on collagen (15Edelman J.M. Chan B.M.C. Uniyal S. Onodera H. Wang D.Z. Damjanovich L. Latzer D.B. Finberg R. Bergelson J.M. Cell Adhesion and Commun. 1994; 2: 131-143Crossref PubMed Scopus (11) Google Scholar), CHO cells transfected with human α2, and mock-transfected CHO cells were previously described (16Bergelson J.M. St. John N. Kawaguchi S. Chan M. Stubdal H. Modlin J. Finberg R.W. J. Virol. 1993; 67: 6847-6852Crossref PubMed Google Scholar). CHO cells expressing human α2 mutants with alanine substitutions at Asp151, Thr221, and Asp254 have also been described (5Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 7Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar). Surface expression of wild-type human α2, and of human α2 with the D151A, T221A, and D254A mutations, was detected by indirect immunofluorescent flow cytometry with monoclonal antibody HAS-4 (20Tenchini M.L. Adams J.C. Gilbert C. Steel J. Hudson D.L. Malcovati M. Watt F.M. Cell Adhesion and Commun. 1993; 1: 55-66Crossref PubMed Scopus (47) Google Scholar) (provided by F. M. Watt) and fluorescein isothiocyanate-conjugated goat antibody to mouse immunoglobulin obtained from Sigma. The negative control antibody MOPC 195 (mouse IgG2b, κ) was obtained from Sigma. Surface expression of wild-type murine α2 was measured using hamster monoclonal antibody HMα2 and the negative control antibody A19-3, with fluorescein isothiocyanate-conjugated mouse antibody to hamster immunoglobulin, all obtained from Pharmingen. Additional monoclonal antibodies were used to test the cell line expressing chimeric murine α2 containing human amino acids 199–216: 12F1 (21Pischel K.D. Hemler M.E. Huang C. Bluestein H.G. Woods V.L. J. Immunol. 1987; 138: 226-233PubMed Google Scholar) was provided by M. Hemler; 5E8 (22Zylstra S. F. A.-Chen Ghosh S.K. Repasky E.A. Rao U. Takita H. Bankert R.B. Cancer Res. 1986; 46: 6446-6451PubMed Google Scholar) and S. V. C. 1991; Scholar) were obtained from the and on Human and has been described (2Bergelson J.M. Shepley M.P. Chan B.M.C. Hemler M.E. Finberg R.W. Science. 1992; 255: 1718-1720Crossref PubMed Scopus (248) Google Scholar). 1 to human not to murine VLA-2 (15Edelman J.M. Chan B.M.C. Uniyal S. Onodera H. Wang D.Z. Damjanovich L. Latzer D.B. Finberg R. Bergelson J.M. Cell Adhesion and Commun. 1994; 2: 131-143Crossref PubMed Scopus (11) Google Scholar), and with human-murine chimeras have that the human I domain is essential for virus binding (4Bergelson J.M. St. John N.F. Kawaguchi S. Pasqualini R. Berdichevsky F. Hemler M.E. Finberg R.W. Cell Adhesion and Commun. 1994; 2: 455-464Crossref PubMed Scopus (51) Google Scholar, S.L. Cunningham J.A. Finberg R.W. Bergelson J.M. J. Virol. 1995; 69: 3237-3239Crossref PubMed Google Scholar). confirm that this depends on within the I domain measured virus attachment to the isolated murine and human I produced as GST fusion proteins GST and a CR3 I domain-GST fusion protein were used as negative previously virus to the human I domain (6King S.L. Cunningham J.A. Finberg R.W. Bergelson J.M. J. Virol. 1995; 69: 3237-3239Crossref PubMed Google Scholar). no virus to the murine I domain, amino acid (15Edelman J.M. Chan B.M.C. Uniyal S. Onodera H. Wang D.Z. Damjanovich L. Latzer D.B. Finberg R. Bergelson J.M. Cell Adhesion and Commun. 1994; 2: 131-143Crossref PubMed Scopus (11) Google Scholar) to the human protein. These results that virus attachment to human VLA-2 depends on sequence between the human and murine I the essential a of chimeric I domains in the human and murine were 2 and to bind virus attachment to human-murine The human VLA-2 I domain is by at the of the The murine VLA-2 I domain is by the at of the of amino acids are by on of the of human Human sequence by murine amino acids are by The number for each is in to the of each Binding of echovirus 1 to each is shown to of each as the of virus to the wild-type human I domain, and is from the of at of human Human sequence introduced into the murine I domain in of murine amino acids are shown in and binding results are shown as described We first a of chimeras in within the human I domain were by murine A on the crystal structure of the CR3 I domain (12Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (806) Google Scholar), was used to of amino acids for that were contained within or sequence virus binding to the human I domain, that the sequence between human and murine I domains were not to any of the of the human I domain had no effect on virus binding. of the portion of the human I domain binding to chimeras sequence chimeras and in amino acids 199–201 and were to bind virus of additional chimeric protein in amino acids 199–216 were all by murine or no virus, that this of the I domain was for virus attachment. In a of murine by human amino acids 199–216 3 the murine I domain into a protein of binding virus of confirm the results obtained with I domain fusion proteins, human amino acids 199–216 were introduced into the murine α2 subunit amino and expressed on the of CHO cells in with Chimeric murine α2 containing human amino acids 199–216 was by the α2 not by or HAS-4 in flow cytometry as previously (4Bergelson J.M. St. John N.F. Kawaguchi S. Pasqualini R. Berdichevsky F. Hemler M.E. Finberg R.W. Cell Adhesion and Commun. 1994; 2: 455-464Crossref PubMed Scopus (51) Google J.M. Chan B.M.C. Uniyal S. Onodera H. Wang D.Z. Damjanovich L. Latzer D.B. Finberg R. Bergelson J.M. Cell Adhesion and Commun. 1994; 2: 131-143Crossref PubMed Scopus (11) Google Scholar), no virus to CHO cells expressing murine α2 virus at to the VLA-2 containing human amino acids 199–216 the results obtained with chimeric I domain fusion proteins were with obtained with chimeric VLA-2 expressed on the cell of monoclonal antibodies with transfected CHO I I I I cytometry was used to of monoclonal antibodies with CHO cell expressing human α2 murine α2 or chimeric murine α2 containing human amino acids 199–216 as as mock-transfected CHO cells The of cells is in a cytometry was used to of monoclonal antibodies with CHO cell expressing human α2 murine α2 or chimeric murine α2 containing human amino acids 199–216 as as mock-transfected CHO cells The of cells is One of the results obtained with chimeras and was that echovirus 1 two with the human I domain, one amino acids and the other amino acids at either of these sites be for virus as for chimeras and not be by of either site test this produced additional chimeras in at each murine were by human of human amino acids 199–201 the murine I domain into a protein of wild-type that these amino acids, of other specific human amino acids, virus attachment. of murine amino acids was to virus binding at of wild-type binding to amino acids was using two additional chimeric I domain containing human amino acids and in the murine protein. with human residues 212–216, echovirus at to the wild-type human I domain with human residues not bind virus. These results confirm that the of human at either of two sites, one residues and the other residues 212–216, is for virus binding to and suggest that virus attachment with each of these VLA-2 interactions with collagen are on divalent and mutations in residues the MIDAS abolish adhesion to collagen. In virus attachment to VLA-2 occurs in the absence of divalent MIDAS residues are between human and murine VLA-2, and be responsible for virus attachment to the as to the I whether the MIDAS residues are for virus measured virus binding to VLA-2 mutants expressed on CHO in the MIDAS residues Asp151, Thr221, and Asp254 were by of these mutations abolishes cell adhesion to collagen (5Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 7Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar). The D151A, T221A, and D254A mutations not inhibit echovirus 1 binding to the transfected CHO cells binding to the mutant cell was to α2 expression as by flow Mutations at Asp254 and not were previously shown to inhibit binding of I domain fusion proteins to collagen (7Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar); of the MIDAS residues had no effect on echovirus 1 binding to isolated I domain-GST fusion proteins These results between the I domain sites required for interaction with collagen and with virus of residues for virus binding to the VLA-2 I of the VLA-2 I domain structure are shown J. S.L. Bergelson J.M. Liddington J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and chain The is in both from one of the in virus attachment are shown and in the The MIDAS residues are shown in and the magnesium ion, in structure are in 1, and and Although the human and murine VLA-2 I domains amino acid echovirus 1 only to the human I In the human-murine chimeric I domains were used to identify responsible for virus attachment to the as to the I These are distinct from the MIDAS residues essential for interactions with collagen, have now shown to be for virus binding. within the human I domain have been identified as for echovirus 1 binding. acids 199–201 and to independently with either site is for virus to and both be by murine to abolish virus binding. Although these results do not the that amino acids between the human and mouse virus, such residues to the of the We have also echovirus 1 binding to additional CHO cell described in 7Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar) in amino acids were by Although α2 expression of these alanine mutants was and not the of in virus no in a amino acid was to abolish virus binding to VLA-2. T. N. St. J. S. and Y. both murine and human VLA-2 bind collagen, it is that the involved in virus attachment are essential for VLA-2 interactions with collagen. mutations within the metal ion-dependent adhesion site of the human VLA-2 I domain, D151A, T221A, and were for on echovirus 1 attachment. of these mutations collagen binding to VLA-2 (5Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 7Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar), not inhibit virus attachment to mutant VLA-2 or isolated I domain-GST fusion (4Bergelson J.M. St. John N.F. Kawaguchi S. Pasqualini R. Berdichevsky F. Hemler M.E. Finberg R.W. Cell Adhesion and Commun. 1994; 2: 455-464Crossref PubMed Scopus (51) Google Scholar, J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar, 15Edelman J.M. Chan B.M.C. Uniyal S. Onodera H. Wang D.Z. Damjanovich L. Latzer D.B. Finberg R. Bergelson J.M. Cell Adhesion and Commun. 1994; 2: 131-143Crossref PubMed Scopus (11) Google Scholar) that the sites for collagen and for echovirus 1 are domain interactions an MIDAS and also residues on a the MIDAS C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S.L. M.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus (51) Google Scholar, Y. M.E. M. J. 1996; PubMed Scopus Google Scholar, L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). has been that the magnesium by the I domain MIDAS in ligand binding by an from a ligand molecule (12Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (806) Google Scholar). 1 binding to the human VLA-2 I domain is in that it does not divalent cations (9Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar) and is not by mutations in the The crystal structure of the human VLA-2 I domain J. S.L. Bergelson J.M. Liddington J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and that residues 199–201 and in two between and and between 3 and the residues are on the of the I domain and are for attachment to virus. The residues on one of the domain, a at one of the also residues from and a viral attachment results do not a for other residues on this that are in murine and human VLA-2. We also that this does not structure in the two of the CR3 I domain J.-O. Arnaout M.A. Liddington Structure. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), with the of virus attachment to (9Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar). The viral attachment is distinct from the MIDAS of the at the of the The two and it is that an that T. Puzon W. Takada Y. J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar), VLA-2 interactions with both virus and collagen (5Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar). on the CR3 I domain that a of the domain at the of the is involved in of ligand binding L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (48) Google Scholar). is also distinct from the viral attachment the two at the The crystal structure of echovirus 1 D. J. J. and J. in has also been and is for on the virus at both the and either of be the site for receptor and either of of the VLA-2 I is that the essential receptor residues identified with viral residues one of the the of both and be required to these interactions in We E. and for N. St. John for F. M. Watt for monoclonal and R. Finberg and M. Hemler for on the