Formation and nature of serpin complexes with serine and cysteine proteinases

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Protein proteinase inhibitors of the serpin superfamily play an important role in regulating intracellular and extracellular proteolytic enzymes in blood coagulation, fibrinolysis, inflammation, apoptosis and other key physiological processes. Serpins are distinguished by their ability to inhibit both serine and cysteine proteinases and by their novel mechanisms of trapping proteinases in kinetically stable covalent complexes through major conformational changes. While the multi-step serpin inhibitory mechanism has provided serpins with function through natural mutations associated with disease. The long range goals of this project are to dissect the stepwise sequence of molecular events involved in the conformational trapping mechanism by which serpins inhibit proteinases and to characterize the molecular events involved in the conformational trapping mechanisms by which serpins inhibit proteinases and to characterize the molecular basis of kinetic stabilization of the complexes for both serine and cysteine proteinase targets. The knowledge gained from such studies is expected to deepen our understanding of how serpins regulate proteolysis and to illuminate the multiple ways in which serpin mutations disrupt this regulation. Three hypotheses for how serpins function as unique protein proteinase inhibitors will be tested in these studies: i) serpins function as suicide substrate inhibitors, being initially recognized as normal substrates by their target proteinase, by then being induced to undergo a major conformational change which traps the proteinase at the acyl-intermediate stage of proteolysis; ii) the trapping of proteinases in stable serpin-proteinase complexes results from conformational changes induced in the proteinase by the serpin which disrupt the proteinase catalytic machinery and thereby prevent deacylation of the complex; iii) cysteine proteinases are inhibited by serpins by the same suicide substrate mechanism of kinetic trapping but with different outcomes dictated by the greater reactivity of the thioester linkage between serpin and proteinase. The three specific aims which will test the hypotheses are: 1) to elucidate the novel multi-step mechanism by which serpins inhibit serine proteinases; 2) to determine the nature of the trapping mechanism and elucidate the role of proteinase conformation changes in inducing the trap; and 3) to determine the mechanism by which serpins inhibit cysteine proteinases and assess any differences from serine proteinase inhibition mechanism.