PROBING SERPIN REACTIVE-LOOP CONFORMATIONS BY PROTEOLYTIC CLEAVAGE

Several crystal structures of intact members of the serine proteinase inhibitor (or serpin) superfamily have recently been solved but the re lationship of their reactive-loop conformations to those of circulatin g forms remains unclear. Here we examine reactive-loop conformational changes of anti-trypsin and antithrombin by using limited proteolysis and binary complex formation with synthetic homologous reactive-loop p eptides. Proteolysis at the P10-P9, P8-P7 and P7-P6 of anti-trypsin wa s distorted by binary complex formation. The P1'-P2' bond in anti-thro mbin 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 id entical with that of the native form and no cleavage was observed in t he hinge region (P15-P10) of either protein, whether native or as bina ry complexes. These results fit with the proposal that the hydrophobic reactive loop of serpins adopts a modified helical conformation in th e circulation, with the hinge region being partly incorporated into th e A beta-pleated sheet. This loop can be displaced by peptides and ind uced to adopt a new conformation similar to the three-turn helix of ov albumin. Both the native and binary complexed forms of anti-thrombin s howed a greatly increased proteolytic sensitivity in the presence of h eparin, indicating that heparin either induces a conformational change in the local structure of the helical reactive loop or facilitates th e approximation of enzyme and inhibitor.