The serpin inhibition mechanism and unique functions of antithrombin.

摘要

Serpins inhibit serine proteinases by a unique mechanism that involves a major conformational change and results in formation of covalent serpin-proteinase complexes. Upon binding to serpin and cleavage of its exposed reactive center loop, covalently-bound proteinase is translocated to the opposite side of the serpin where it is crashed against the rigid serpin body and kept catalytically inactive due to the distortion of its active site. Whereas this had been shown to be the case for alpha1PI-trypsin pair, as demonstrated by the X-ray structure of the covalent complex, it was not clear if other serpins form complexes of the similar structural organization, and if this depends on the nature of the proteinase involved. To answer this question the study of PAI-1 and four proteinases of different size and domain organization was conducted. The results demonstrate that all four covalent complexes have the same structural organization, the one resembling the covalent complex of alpha 1PI and trypsin. The similarity in the structures of the covalent complexes is indicative of a common inhibitory mechanism that alpha1PI and PAI-1 utilize to inhibit proteinases.; It was suggested that the structure of proteinase moiety is altered when proteinase is complexed to serpin. To characterize the nature of the conformational change we employed NMR as a sensitive, solution and non-perturbing method. Trypsin was uniformly labelled with 15N and [15N- 1H]-HSQC spectra were recorded for native trypsin and trypsin in covalent complex with alpha1PI. The results indicate a massive change in trypsin conformation that possibly involves a loss of secondary or tertiary structures.; To apply NMR to serpins, enrichment with NMR sensitive nuclei is required. Protocols for labelling of alpha1PI and PAI-1 with 15 N-valine, antithrombin with 15N-alanine and alpha 1PI with 15N, 13C and 2H were developed and will allow the application of NMR to the studies of these serpins.; Antithrombin is a principal inhibitor of thrombin and factor Xa and a serpin that requires heparin for its unique inhibition mechanism. Determination of three new antithrombin sequences and the detailed analysis of the antithrombin conservation is presented. Sequence comparisons of the new sequences with the other 10 previously known antithrombin sequences, and with sequences of other serpins provided striking evidence for the conservation of the heparin activation mechanism, and new insight into the residues important for heparin binding, heparin activation, as well as an indication of which glycosylation sites might be needed for function. A potential factor Xa exosite needed for enhancement of factor Xa inhibition and residues important in expressing the anti-angiogenic properties of antithrombin have been proposed based on the conservation data.