title: Divalent cations stabilize the alpha 1 beta 1 integrin I domain.
abstract: Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.
authors.affiliation: Biogen, Inc., Cambridge, Massachusetts 02142, USA. philip_gotwals@biogen.com
authors.forename: P J
authors.initials: PJ
authors.lastname: Gotwals
authors.forename: G
authors.initials: G
authors.lastname: Chi-Rosso
authors.forename: S T
authors.initials: ST
authors.lastname: Ryan
authors.forename: I
authors.initials: I
authors.lastname: Sizing
authors.forename: M
authors.initials: M
authors.lastname: Zafari
authors.forename: C
authors.initials: C
authors.lastname: Benjamin
authors.forename: J
authors.initials: J
authors.lastname: Singh
authors.forename: S Y
authors.initials: SY
authors.lastname: Venyaminov
authors.forename: R B
authors.initials: RB
authors.lastname: Pepinsky
authors.forename: V
authors.initials: V
authors.lastname: Koteliansky
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D000915:Antibody Affinity; D000918:Antibody Specificity; D002413:Cations, Divalent / Q000737:chemistry*; D003094:Collagen / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D039222:Integrin alpha1beta1; D016023:Integrins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008274:Magnesium / Q000737:chemistry / Q000502:physiology*; D008345:Manganese / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D000939:Epitopes; D039222:Integrin alpha1beta1; D016023:Integrins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D008345:Manganese; D003094:Collagen; D008274:Magnesium