Characterization of a Site on PAI-1 That Binds to Vitronectin Outside of the Somatomedin B Domain

• Published in: The Journal of biological chemistry 2008-10, Vol.283 (42), p.28487-28496
• Main Authors: Schar, Christine R., Jensen, Jan K., Christensen, Anni, Blouse, Grant E., Andreasen, Peter A., Peterson, Cynthia B.
• Format: Article
• Language: English
• Subjects: Binding Sites; Humans; Kinetics; Models, Biological; Models, Chemical; Mutagenesis, Site-Directed; Mutation; Plasminogen Activator Inhibitor 1 - chemistry; Protein Binding; Protein Conformation; Protein Structure and Folding; Protein Structure, Tertiary; Somatomedins - chemistry; Temperature; Time Factors; Vitronectin - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M804257200

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are proteins that interact in the circulatory system and pericellular region to regulate fibrinolysis, cell adhesion, and migration. The interactions between the two proteins have been attributed primarily to binding of the somatomedin B (SMB) domain, which comprises the N-terminal 44 residues of vitronectin, to the flexible joint region of PAI-1, including residues Arg-103, Met-112, and Gln-125 of PAI-1. A strategy for deletion mutagenesis that removes the SMB domain demonstrates that this mutant form of vitronectin retains PAI-1 binding (Schar, C. R., Blouse, G. E., Minor, K. M., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297–10309). In the current study, the complementary binding site on PAI-1 was mapped by testing for the ability of a battery of PAI-1 mutants to bind to the engineered vitronectin lacking the SMB domain. This approach identified a second, separate site for interaction between vitronectin and PAI-1. The binding of PAI-1 to this site was defined by a set of mutations in PAI-1 distinct from the mutations that disrupt binding to the SMB domain. Using the mutations in PAI-1 to map the second site suggested interactions between α-helices D and E in PAI-1 and a site in vitronectin outside of the SMB domain. The affinity of this second interaction exhibited a KD value ∼100-fold higher than that of the PAI-1-somatomedin B interaction. In contrast to the PAI-1-somatomedin B binding, the second interaction had almost the same affinity for active and latent PAI-1. We hypothesize that, together, the two sites form an extended binding area that may promote assembly of higher order vitronectin-PAI-1 complexes.