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

Abstract

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/?-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 B-sheet A in the cleaved form.

Antithrombin‐TRI (Ala 382 to Thr) causing severe thromboembolic tendency undergoes the S‐to‐R transition and is associated with a plasma‐inactive high‐molecular‐weight complex of aggregated antithrombin

“…Secondly, our present study and an earlier report (Lindo et al, 1993) showed that on cleavage of the reactive centre loop of AT-TRI (P12 variant) a large increase in thermostability occurs, characteristic of the S-to-R transition (this has also recently been reported by Wright & Blajchman, 1994). In spite of the capacity of antithrombin-TRI to undergo this transformation, however, its cleaved form did not react with a monoclonal antibody, 4C9, possibly indicating that a modified S-to-R transition had occurred.…”

The Contribution of the Conserved Hinge Region Residues of .alpha.1-Antitrypsin to Its Reaction with Elastase

“…It is possible that the assay used here does not identify subtle changes in structure that the immunological methods of Skriver et al (1991) recognized. Nevertheless, in conjunction with other studies (Hood et al, 1994;Hopkins et al, 1993;Lawrence et al, 1994;Wright & Blajchman, 1994), our results demonstrate that cleaved hinge region variants still undergo a dramatic increase in stability upon cleavage, and that small amino acids in the hinge region are not an absolute requirement for the S to R transition. Examination of cleaved serpin structures (Baumann et al, 1991;Delarue et al, 1990; Figure 4: Analysis of the reaction products arising from the incubation of hinge region variants with elastase by 10% acrylamide SDS-PAGE.…”

Engineering an Anion-Binding Cavity in Antichymotrypsin Modulates the “Spring-Loaded” Serpin−Protease Interaction

“…The P10-P14 residues serve as a conformational hinge for the serpin rearrangement illustrated in Figure 1; the P-even residues (P10, P12, P14) are ultimately buried in the hydrophobic protein core. Although it had been considered that radical mutations of P-even hinge residues would inhibit strand s4A insertion and therefore block inhibitory activity (42,43), these variants show increased stability upon reactive loop cleavage, suggesting at least some degree of strand s4A insertion (33,34,39,44). This suggestion has been verified by recent X-ray crystal structure determinations of cleaved antichymotrypsin variants with P14 and P12 arginine substitutions (45) and a cleaved plasminogen activator inhibitor with a proline residue substituted at the P12 position (38).…”