Interactions of Plasminogen Activator Inhibitor-1 with Vitronectin Involve an Extensive Binding Surface and Induce Mutual Conformational Rearrangements

Abstract

In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into beta-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496).

Metals affect the structure and activity of human plasminogen activator inhibitor‐1. I. Modulation of stability and protease inhibition

“…Second, the PAI‐1/SMB complex in the presence of CuCl 2 is somewhat less stable than the PAI‐1/vitronectin complex in the presence of CuCl 2. These differences are consistent with a relatively minor contribution from the more extensive PAI‐1/vitronectin interaction surface that extends beyond the sequence encompassed by the SMB domain 35–37. Aside from these nuances, data on the complexes with the SMB domain largely recapitulate the metal effects on PAI‐1 observed with full‐length vitronectin.…”

“…A considerable body of work recently published by our group35–37 has established a more extensive binding interface between vitronectin and PAI‐1 than had previously been recognized. These studies have shown that binding sites exist in addition to the well‐characterized interactions between PAI‐1 the N‐terminal SMB domain of vitronectin 35–37. Therefore, we were interested to see whether the same dramatic effects on metal modulation of PAI‐1 stability observed with full‐length vitronectin were exhibited by the SMB domain.…”

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

“…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.…”

Elucidation of the Contribution of Active Site and Exosite Interactions to Affinity and Specificity of Peptidylic Serine Protease Inhibitors Using Non-Natural Arginine Analogs

“…Although a number of inhibitors of human uPA have been developed, the species specificity has been studied in only a few cases. Species-specific inhibitory polyclonal and monoclonal antibodies (Danø et al, 1980;Kaltoft et al, 1982;Petersen et al, 2001;Lund et al, 2008;Blouse et al, 2009) are available but lack some of the advantageous properties of peptides. Some aryl-amidine-based, small molecule inhibitors had a strong preference (up to 203-fold) for human uPA over murine uPA (Klinghofer et al, 2001).…”