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

Abstract

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 ␣-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 K D value ϳ100-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.Vitronectin is a circulatory protein that interacts with several other plasma components that regulate coagulation and fibrinolysis, cell lysis via the complement cascade, and cell binding and migration in an integrin-dependent and urokinase-type plasminogen activator receptor-dependent fashion (1, 2). An interaction with vitronectin that has been widely studied occurs with the serine protease inhibitor (or serpin), PAI-1, 2 the primary inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Most circulating PAI-1 is found in a complex with vitronectin (3). The formation of the complex between vitronectin and PAI-1 is presumed to be physiologically significant in several respects. Binding of vitronectin to PAI-1 stabilizes the active conformation of the serpin, increasing the half-life for its conversion to the inactive, so-called latent state, and maintains its protease inhibitory properties for a prolonged period (4, 5). PAI-1 binds to the N-terminal ϳ50-amino acid somatomedin B (SMB) domain of vitronectin. The binding of PAI-1 competes with the binding of the urokinase-type plasminogen activator receptor to this domain and with the binding of integrins to the adjacent RGD sequence (for a review, see Ref. 6). Moreover, PAI-1 has been reported to induce oligomerization of vitronectin (7-10).Th...

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

“…9). There is also evidence that VN has additional binding interactions with active PAI-1 outside the somatomedin domain B involving residues in ␣-hD (Lys-69, Arg-76, Tyr-79, and Lys-80) and ␣-hE (Arg-115 and Arg-118) (26,27). Some of these residues are the same as those interacting with AZ3976 in the present structure, i.e.…”

“…Still, PAI-1 ex vivo in blood has a half-life of only ϳ1 h at 37°C (24). It has been found that PAI-1 binds to the N-terminal ϳ50-amino acid somatomedin B domain of VN (25)(26)(27). X-ray evidence (23) suggests that binding to this domain induces conformational changes in PAI-1 which, in addition to steric effects, may contribute to its improved stability.…”

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

“…DENV NS1-VN interaction is increased by PAI-1 and inhibited by plasminogen. VN can bind several molecules, such as heparin, PAI-1, HPX, and PLG, and these interactions occur at distinct sites in the VN molecule (23)(24)(25)(26)(27). Therefore, to identify the putative binding sites of NS1 on the VN molecule, competitive ELISAs were performed in which VN ligands were used as competitors (28).…”

Inhibition of neutrophil apoptosis by PAI-1

“…Plasminogen activator inhibitor 1 (PAI-1) is an effective inhibitor of plasminogen activators, particularly urokinase-plasminogen activator (uPA) and tissue-type plasminogen activator, and binds to the LDL receptor (LRP) and vitronectin (25,33). Although increased levels of PAI-1 and inhibition of plasmin generation are found in the lungs and circulation of critically ill patients with sepsis or ALI (28,40), recent studies have also shown that PAI-1 can affect a broad range of cellular functions independent of its effects on coagulation, including cell adhesion, proliferation, migration, and viability (6,21,36).…”