INTERFERING WITH THE INHIBITORY MECHANISM OF SERPINS - CRYSTAL-STRUCTURE OF A COMPLEX FORMED BETWEEN CLEAVED PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 AND A REACTIVE-CENTER LOOP PEPTIDE

Background: Plasminogen activator inhibitor type I (PAI-I) is an impor tant endogenous regulator of the fibrinolytic system. Reduction of PAI -1 activity has been shown to enhance dissolution of blood clots. Like other serpins, PAI-1 binds covalently to a target serine protease, th ereby irreversibly inactivating the enzyme. During this process the ex posed reactive-centre loop of PAI-1 is believed to undergo a conformat ional change becoming inserted into beta sheet A of the serpin. Incuba tion with peptides from the reactive-centre loop transform serpins int o a substrate for their target protease. It has been hypothesised that these peptides bind to beta sheet A, thereby hindering the conformati onal rearrangement leading to loop insertion and formation of the stab le serpin-protease complex, Results: We report here the 1.95 Angstrom X-ray crystal structure of a complex of a glycosylated mutant of PAI-1 , PAI-1-Ala335Glu, with two molecules of the inhibitory reactive-centr e loop peptide N-Ac-TVASS-NH2. Both bound peptide molecules are locate d between beta strands 3A and 5A of the serpin. The binding kinetics o f the peptide inhibitor to immobilised PAI-1-Ala335Glu, as monitored b y surface plasmon resonance, is consistent with there being two differ ent binding sites. Conclusions: This is the first reported crystal str ucture of a complex formed between a serpin and a serpin inhibitor, Th e localisation of the inhibitory peptide in the complex strongly suppo rts the theory that molecules binding in the space between beta strand s 3A and 5A of a serpin are able to prevent insertion of the reactive- centre loop into beta sheet a, thereby abolishing the ability of the s erpin to irreversibly inactivate its target enzyme. The characterisati on of the two binding sites for the peptide inhibitor provides a solid foundation far computer-aided design of novel, low molecular weight P AI-1 inhibitors.