Effect of stabilizing versus destabilizing interactions on plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin family, as it spontaneously converts into a latent conformation. However, t he exact mechanism of this conversion is not known. Previous studies report ed that neutralizing monoclonal antibodies as well as reversal or removal o f charges on the s3C-s4C turn results in a destabilization of PAI-1 leading to an accelerated conversion to its latent form. In this study the effect of the reversal or removal of charges in this "gat e region" (R186E/R187E, H190E/K191E, H190L/K191L and R356E) on a stable PAI -l-variant (PAI-l-stab) was investigated. Whereas PAI-l-stab has a half-lif e of 150 +/- 66 h, PAI-1-stab-R186E-R187E, PAI-1-stab-H190E-K191E, PAI-1-sr ab-H190L-K191L and PAI-1-stab-R356E have a strongly decreased half-life (p < 0.005 versus PAI-1-stab) of 175 +/- 48 min, 75 +/- 34 min, 68 +/- 38 min and 79 +/- 16 min, respectively. Wild-type PAI-1 (wtPAI-1) had a half-life of 55 +/- 19 min. These data indicate that the stabilization induced by the mutated residues in PAI-1-stab is counteracted by the additional mutations , resulting in half-lives similar to that of wtPAI-1, thereby suggesting th at the stabilizing and destabilizing forces act mainly independently in the se mutants. Extrapolation of these data to other (stable) serpins leads to the hypothesis that the s3C-s4C turn and the distal hinge region of the rea ctive site loop plays a role for the stability of serpins in general.