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

Christine R Schar,Jan K Jensen,Anni Christensen,Grant E Blouse,Peter A Andreasen,Cynthia B Peterson

doi:10.1074/jbc.m804257200

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 alpha-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 approximately 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.

Highlights

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]
Where is the second site located relative to the flexible joint region of plasminogen activator inhibitor-1 (PAI-1)? Does binding of vitronectin to this site stabilize the active conformation of PAI-1? How do the kinetics of binding to the second site compare with the binding to the somatomedin B (SMB) domain?
The Deletion Mutant of Vitronectin Lacking the SMB Domain Does Not Stabilize the Active Conformation of PAI-1—One of the key consequences of vitronectin binding to PAI-1 is stabilization of the active serpin, with its reactive center loop (RCL) exposed and poised to act as bait for target proteases

Summary

EXPERIMENTAL PROCEDURES

Materials—Native vitronectin was purified from human plasma using a modified protocol of the method developed by Dahlback and Podack [33, 34]. To determine the affinity of PAI-1 binding to full-length vitronectin or r⌬sBVN, samples of PAI-1 diluted in running buffer were injected into an array of concentrations ranging from 0.25 to 1500 nM at a flow rate of 30 ␮l/min. The kon values were determined by following the association phase at different PAI-1 concentrations. The apparent association rate constants (kon,app) were determined by a fit (global with the Biacore software package or individual curve first for the manual data treatment) to Equation 1,. In some cases the association and dissociation rates were too fast to be determined In these cases, the KD values were calculated according to the method outlined by Rich and Myszka [36] for use of SPR in an equilibrium binding approach. The steady state binding values were plotted against the PAI-1 concentration and fit by nonlinear least squares to the equation,

KD C

RESULTS

Wild type