Proteolytically cleaved mutant antithrombin-Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins.

The serpin family of proteins consists primarily of proteinase inhibitors which form tight complexes with target proteinases. Inhibitor serpins are cleaved by proteinase and undergo a large conformational change in which the polypeptide segment terminating at the target reactive site, at which cleavage takes place, inserts itself as an additional strand, s4A, in the center of a preexisting beta-sheet. This change in conformation increases the stability towards denaturation of the cleaved serpin relative to the native uncleaved form. Mutant serpins with single amino acid changes in the s4A strand have been identified, and in most cases these are proteinase substrates but not inhibitors. We have measured the stability to denaturation of one of these non-inhibitor substrate mutants, antithrombin-Hamilton, which has an Ala-->Thr change at position P12 in strand s4A. We find that it undergoes the transformation to the more stable form which is observed for inhibitor serpins, from which we conclude that the Ala-->Thr change in antithrombin-Hamilton does not prevent insertion of s4A into beta-sheet A in the cleaved form.