Structure of a serpin-enzyme complex probed by cysteine substitutions and fluorescence spectroscopy

The x-ray crystal structure of the serpin-proteinase complex is yet to be d etermined. In this study we have investigated the conformational changes th at take place within antitrypsin during complex formation with catalyticall y inactive (thrombin(S195A)) and active thrombin. Three variants of antitry psin Pittsburgh (an effective thrombin inhibitor), each containing a unique cysteine residue (Cys(232), Cys(P3'), and Cys(313)) were covalently modifi ed with the fluorescence probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz -2-oxa-1,3-diazol-4-yl)ethylenediamine. The presence of the fluorescent lab el did not affect the structure or inhibitory activity of the serpin. We mo nitored the changes in the fluorescence emission spectra of each labeled se rpin in the native and cleaved state, and in complex with active and inacti ve thrombin. These data show that the serpin undergoes conformational chang e upon forming a complex with either active or inactive proteinase. Steady- state fluorescence quenching measurements using potassium iodide were used to further probe the nature and extent of this conformational change. A pro nounced conformational change is observed upon locking with an active prote inase; however, our data reveal that docking with the inactive proteinase t hrombin(S195A) is also able to induce a conformational change in the serpin .