J Biol Chem. 1997 May; 272(19): 12662-6.
Proteinase specificity and functional diversity in point mutants of plasminogen activator inhibitor 1.
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. The alternative behavior of PAI-1 as an inhibitor, a non-inhibitory substrate, or a non-reactive latent form has been shown to be dependent on the initial conformation. In this study, we have evaluated the effect of a substitution outside the reactive site loop (P18) or in the reactive site loop (P6 and P10) on proteinase specificity and conformational transitions in PAI-1. Wild-type PAI-1 (wtPAI-1) revealed the same conformational distribution pattern toward tissue-type plasminogen activator (t-PA) as toward urokinase-type plasminogen activator (u-PA) (i.e. 53 +/- 6. 9% active, 36 +/- 6.8% latent, and 12 +/- 1.9% substrate). Inactivation of wtPAI-1 resulted in the conversion of the labile active form into the latent form while the stable substrate form remained unchanged. PAI-1-P6 (Val --> Pro at P6) revealed a target specificity for t-PA (39 +/- 7% versus 3 +/- 2% of the theoretical maximal value toward t-PA and u-PA, respectively), PAI-1-P10 (Ser --> Pro at P10) was 4-fold more active toward u-PA than toward t-PA, and PAI-1-P18 (Asn --> Pro at P18) exhibited inhibitory properties exclusively toward u-PA (41 +/- 10%). Surprisingly, inactivation of these mutants revealed functional and conformational transitions distinct from those observed for wtPAI-1. Inactivation of PAI-1-P6(Val --> Pro) resulted in a total conversion of the active form into the latent form and in a partial conversion of the substrate form into the latent form. The active forms of both PAI-1-P10(Ser --> Pro) and PAI-1-P18(Asn --> Pro) are also labile but, in contrast to the active form of wtPAI-1, convert into substrate forms. Based on the existence of various conformations of PAI-1, we propose an alternative reaction scheme describing the putative interactions between serpins and their target proteinases. The unusual conformational and functional flexibility of PAI-1 that, according to the current study, appears not to be restricted to the reactive site loop further underlines the importance of potential structural rearrangements (e.g. upon binding to cofactors) in PAI-1 (or serpins in general) for its functional behavior at particular biological sites.