Characterization of the binding of different conformational forms of plasminogen activator inhibitor-1 to vitronectin. Implications for the regulation of pericellular proteolysis.
Plasminogen activator inhibitor type 1 (PAI-1), the primary physiologic inhibitor of plasminogen activation, is associated with the adhesive glycoprotein vitronectin (Vn) in plasma and the extracellular matrix. In this study we examined the binding of different conformational forms of PAI-1 to both native and urea-purified vitronectin using a solid-phase binding assay. These results demonstrate that active PAI-1 binds to urea-purified Vn with approximately 6-fold higher affinity than to native Vn. In contrast, inactive forms of PAI-1 (latent, elastase-cleaved, synthetic reactive center loop peptide-annealed, or complexed to plasminogen activators) display greatly reduced affinities for both forms of adsorbed Vn, with relative affinities reduced by more than 2 orders of magnitude. Structurally, these inactive conformations all differ from active PAI-1 by insertion of an additional strand into beta-sheet A, suggesting that it is the rearrangement of sheet A that results in reduced Vn affinity. This is supported by the observation that PAI-1 associated with beta-anhydrotrypsin, which does not undergo rearrangement of beta-sheet A, shows no such decrease in affinity, whereas PAI-1 complexed to beta-trypsin, which does undergo sheet A rearrangement, displays reduced affinity for Vn similar to PAI-1.plasminogen activator complexes. Together these data demonstrate that the interaction between PAI-1 and Vn depends on the conformational state of both proteins and suggest that the Vn binding site on PAI-1 is sensitive to structural changes associated with loss of inhibitory activity.
Lawrence
D A
DA
Department of Biochemistry, American Red Cross Holland Laboratory, Rockville, Maryland 20855, USA. lawrencd@usa.redcross.org
Palaniappan
S
S
Stefansson
S
S
Olson
S T
ST
Francis-Chmura
A M
AM
Shore
J D
JD
Ginsburg
D
D
D006868:Hydrolysis; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D019096:Vitronectin / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin