STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE PLASMINOGEN-ACTIVATOR INHIBITOR-1 BINDING MOTIF IN THE SOMATOMEDIN-B DOMAIN OF VITRONECTIN

Plasminogen activator inhibitor 1 (PAI-1) binds to the somatomedin B ( SMB) domain of vitronectin (VN), a domain present in at least seven ot her proteins. In this study, we investigate the PAI-1 binding activity of these SMB homologs and attempt to more specifically localize the P AI-1 binding site within this domain. SMB(VN) and several of its homol ogs were expressed in Escherichia coli, purified, and tested for PAI-1 binding activity in a competitive ligand binding assay. Although reco mbinant SMB(VN) was fully active in this assay, none of the homologs b ound to PAT-1 or competed with VN for PAI-1 binding. These inactive ho mologs are structurally related to SMB(VN), having 33-45% sequence ide ntity and containing all 8 cysteines at conserved positions. Thus, hom olog-scanning experiments were conducted by exchanging progressively l arger portions of the NH2- or COOH-terminal regions of active SMB(VN) with the corresponding regions of the inactive homologs. These experim ents revealed that the minimum PAI-1-binding sequence was present in t he central region (residues 12-30) of SMB(VN). Alanine scanning mutage nesis further demonstrated that each of the 8 cysteines as well as Gly (12), Asp(22), Leu(24), Try(27), Tyr(28) and Asp(34) were critical for PAI-1 binding and were required to stabilize PAI-1 activity. These re sults indicate that the PAI-1 binding motif is localized to residues 1 2-30 of SMB(VN) and suggest that this motif is anchored in the active conformation by disulfide bonds.