Probing serpin reactive-loop conformations by proteolytic cleavage

Abstract

Several crystal structures of intact members of the serine proteinase inhibitor (or serpin) superfamily have recently been solved but the relationship of their reactive-loop conformations to those of circulating forms remains unclear. Here we examine reactive-loop conformational changes of anti-trypsin and anti-thrombin by using limited proteolysis and binary complex formation with synthetic homologous reactive-loop peptides. Proteolysis at the P10–P9, P8–P7 and P7–P6 of anti-trypsin was distorted by binary complex formation. The P1´–P2´ bond in anti-thrombin was more accessible to proteolysis after binary complex formation, whereas cleavage at the P4–P3 bond was variably altered by synthetic peptide insertion. The proteolytic accessibility of the reactive-site P1–P1´ bond of anti-trypsin and anti-thrombin binary complexes was identical with that of the native form and no cleavage was observed in the hinge region (P15–P10) of either protein, whether native or as binary complexes. These results fit with the proposal that the hydrophobic reactive loop of serpins adopts a modified helical conformation in the circulation, with the hinge region being partly incorporated into the A β-pleated sheet. This loop can be displaced by peptides and induced to adopt a new conformation similar to the three-turn helix of ovalbumin. Both the native and binary complexed forms of anti-thrombin showed a greatly increased proteolytic sensitivity in the presence of heparin, indicating that heparin either induces a conformational change in the local structure of the helical reactive loop or facilitates the approximation of enzyme and inhibitor.