The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites

Background: The inhibitors that belong to the serpin family are widely dist ributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-ce ntre cleavage, loop insertion and protease translocation, but different mod els 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 me asurements, based on donor-donor energy migration (DDEM), to accurately tri angulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PA I-1). The distances from residue 344 (P3) in the reactive-centre loop of PA I-1 to residues 185, 266, 313 and 347 (P1') were determined. Modelling of t he complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the rea ctive-centre loop fully inserted into beta 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 measuremen ts of fluorophores attached to these residues and cross-linking using a sul phydryl-specific cross-linker. Conclusions: The data clearly demonstrate that serpin inhibition involves r eactive-centre cleavage followed by full-loop insertion whereby the covalen tly linked protease is translocated from one pole of the inhibitor to the o pposite one.