A method for defining binding sites involved in protein-protein interactions: analysis of the binding of plasminogen activator inhibitor 1 to the somatomedin domain of vitronectin.
Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12-30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24-30) in the SMB domain.
Royle
G
G
Department of Vascular Biology/VB-3, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Deng
G
G
Seiffert
D
D
Loskutoff
D J
DJ
D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D016296:Mutagenesis; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013002:Somatomedins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019096:Vitronectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D013002:Somatomedins; D019096:Vitronectin; D000409:Alanine