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, the exact mechanism of this conversion is not known. Previous studies reported that neutralizing monoclonal antibodies as well as reversal or removal of 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 "gate region" (R186E/R187E, H190E/K191E, H190L/K191L and R356E) on a stable PAI-1-variant (PAI-1-stab) was investigated. Whereas PAI-1-stab has a half-life of 150+/-66 h, PAI-1-stab-R186E-R187E, PAI-1-stab-H190E-K191E, PAI-1-stab-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 that the stabilizing and destabilizing forces act mainly independently in these 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 reactive site loop plays a role for the stability of serpins in general.
Vleugels
N
N
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
Leys
J
J
Knockaert
I
I
Declerck
P J
PJ
D006801:Humans; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation
D017395:Plasminogen Activator Inhibitor 1