The importance of helix F in plasminogen activator inhibitor-1.
Plasminogen activator inhibitor-1 (PAI-1) is the only functionally labile serpin, as it converts spontaneously into a non-reactive 'latent' conformation. Several studies have suggested an important role for helix F in the functional behavior and stability of the serpins, especially for PAI-1. We constructed a mutant of PAI-1 (PAI-1-delhF) in which residues 127-158 (hF-thFs3A) were deleted. Whereas wild-type PAI-1 (wtPAI-1) exhibits inhibitory properties towards t-PA and u-PA to an extent of 60-80% of the theoretical maximum, PAI-1-delhF did not exert any detectable inhibitory properties, but behaved as a stable substrate. Prolonged incubation at 37 degrees C did not change its functional properties in contrast to wtPAI-1 that under those conditions 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 decreased binding to vitronectin. The obtained results clearly show the importance 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 inability to form a stable covalent complex. Moreover, removal of helix F strongly affects the binding of PAI-1 to vitronectin.
Vleugels
N
N
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Van Evenstraat 4, B-3000, Leuven, Belgium.
Gils
A
A
Bijnens
A P
AP
Knockaert
I
I
Declerck
P J
PJ
D000939:Epitopes / Q000737:chemistry; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000235:genetics*; D011489:Protein Denaturation; D010959:Tissue Plasminogen Activator / Q000494:pharmacology; D019096:Vitronectin / Q000737:chemistry
D000939:Epitopes; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D010959:Tissue Plasminogen Activator