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Structure. 2000 Apr 15;8(4):397-405.

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

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  • 1Department of Medical Biosciences, Medical Biochemistry, Umeâ University, Umeâ, S-90187, Sweden.

Abstract

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 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 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.

PMID:
10801484
[PubMed - indexed for MEDLINE]
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