The importance of helix F in plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is the only functionally labile s erpin, as it converts spontaneously into a non-reactive 'latent' conformati on. Several studies have suggested an important role for helix F in the fun ctional behavior and stability of the serpins, especially for PAI-1. We con structed a mutant of PAI-1 (PAI-1-delhF) in which residues 127-158 (hF-thFs 3A) were deleted. Whereas wild-type PAI-1 (mtPAI-1) exhibits inhibitory pro perties towards t-PA and u-PA to an extent of 60-80% of the theoretical max imum, PAI-1-delhF did not exert any detectable inhibitory properties, but b ehaved as a stable substrate. Prolonged incubation at 37 degrees C did not change its functional properties in contrast to wtPAI-1 that under those co nditions converts to the latent conformation. In contrast to active wtPAI-1 and other substrate-type PAI-1 mutants, PAI-1-delhF showed a 3000-fold dec reased binding to vitronectin. The obtained results clearly show the import ance of helix F in the inhibitory activity of PAI-1. The absence of helix F apparently leads to an impaired kinetics of insertion of the reactive site loop upon interaction with its target proteinase resulting in the inabilit y to form a stable covalent complex. Moreover, removal of helix F strongly affects the binding of PAI-1 to vitronectin. (C) 2000 Elsevier Science B.V. All rights reserved.