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 o f this site by employing a novel approach that is based on the use of monoc lonal antibodies (Mabs) together with site-directed mutagenesis. Six separa te Mabs were identified that bound to the SAM 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 sc anning mutagenesis), was then determined and compared in competitive bindin g experiments. Three separate, partially overlapping Mab epitopes within SM B were defined by these studies, and the PAI-1 binding site was localized t o 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 th e SMB domain. (C) 2001 Academic Press.