Mutational analysis of plasminogen activator inhibitor-1.
The serpin plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the plasminogen activating serine proteases tissue-type and urokinase-type plasminogen activator and, as such, an important regulator in turnover of extracellular matrix and in fibrinolysis. PAI-1 spontaneously loses its antiproteolytic activity by inserting its reactive centre loop (RCL) as strand 4 in beta-sheet A, thereby converting to the so-called latent state. We have investigated the importance of the amino acid sequence of alpha-helix F (hF) and the connecting loop to s3A (hF/s3A-loop) for the rate of latency transition. We grafted regions of the hF/s3A-loop from antithrombin III and alpha1-protease inhibitor onto PAI-1, creating eight variants, and found that one of these reversions towards the serpin consensus decreased the rate of latency transition. We prepared 28 PAI-1 variants with individual residues in hF and beta-sheet A replaced by an alanine. We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case. Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour, suggesting that insertion of the RCL is held back during latency transition as well as during complex formation with target proteases. The data presented identify new determinants of PAI-1 latency transition and provide general insight into the characteristic loop-sheet interactions in serpins.
Wind
Troels
T
Laboratory of Cellular Protein Science, Department of Molecular Biology, Aarhus University, Denmark.
Jensen
Jan K
JK
Dupont
Daniel M
DM
Kulig
Paulina
P
Andreasen
Peter A
PA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator