The structure of a serpin–protease complex revealed by intramolecular distance measurements using donor–donor energy migration and mapping of interaction sites
2000 (English)In: Structure, Vol. 8, no 4, 397-405 p.Article in journal (Refereed) Published
Background: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin–protease complex have been proposed. In the absence of a spatial structure of a serpin–protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial.
Results: We used a recently developed method for making precise distance measurements, based on donor–donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1′) were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into β sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease–inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker.
Conclusions: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.
Place, publisher, year, edition, pages
2000. Vol. 8, no 4, 397-405 p.
Cross-linking, Donor–donor energy migration, Fluorescence, Intramolecular distance, PAI-1, serpin
IdentifiersURN: urn:nbn:se:umu:diva-8572DOI: doi:10.1016/S0969-2126(00)00121-0OAI: oai:DiVA.org:umu-8572DiVA: diva2:148243