title: Allosteric beta1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain.
abstract: The ligand binding function of integrins can be modulated by various monoclonal antibodies by both direct and indirect mechanisms. We have characterized an anti-beta(1) antibody, SG/19, that had been reported to inhibit the function of the beta(1) integrin on the cell surface. SG/19 recognized the wild type beta(1) subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant beta(1) integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr(82) in the beta(1) subunit, located at the outer face of the boundary between the I-like and hybrid domains, was the key binding determinant for this antibody. Direct visualization of the alpha (5)beta(1) headpiece fragment in complex with SG/19 Fab with electron microscopy confirmed the location of the binding surface and showed that the ligand binding site is not occluded by the bound Fab. Surface plasmon resonance showed that alpha (5)beta(1) integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-alpha(5) antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-beta antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain without directly interfering with ligand docking.
authors.affiliation: The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Bing-Hao
authors.initials: BH
authors.lastname: Luo
authors.forename: Konstantin
authors.initials: K
authors.lastname: Strokovich
authors.forename: Thomas
authors.initials: T
authors.lastname: Walz
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Springer
authors.forename: Junichi
authors.initials: J
authors.lastname: Takagi
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D019012:Integrin beta1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D019012:Integrin beta1; D021122:Protein Subunits; D011994:Recombinant Proteins