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Biophys J, January 2001, p. 491-497, Vol. 80, No. 1

Structure of a Serpin-Enzyme Complex Probed by Cysteine Substitutions and Fluorescence Spectroscopy

Justin P. Ludeman,* James C. Whisstock,* Paul C.R. Hopkins,dagger Bernard F. Le Bonniec,Dagger and Stephen P. Bottomley*

 *Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia;  dagger Gladstone Institute of Cardiovascular Disease, San Francisco, California 94141 USA;  Dagger INSERM, Unité 428, Univeristé Paris V, 75270 Paris, France

The x-ray crystal structure of the serpin-proteinase complex is yet to be determined. In this study we have investigated the conformational changes that take place within antitrypsin during complex formation with catalytically inactive (thrombinS195A) and active thrombin. Three variants of antitrypsin Pittsburgh (an effective thrombin inhibitor), each containing a unique cysteine residue (Cys232, CysP3', and Cys313) were covalently modified 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 label did not affect the structure or inhibitory activity of the serpin. We monitored the changes in the fluorescence emission spectra of each labeled serpin in the native and cleaved state, and in complex with active and inactive thrombin. These data show that the serpin undergoes conformational change 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 pronounced conformational change is observed upon locking with an active proteinase; however, our data reveal that docking with the inactive proteinase thrombinS195A is also able to induce a conformational change in the serpin.

Biophys J, January 2001, p. 491-497, Vol. 80, No. 1
© 2001 by the Biophysical Society   0006-3495/01/01/491/07  $2.00


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P. Mellet, Y. Mely, L. Hedstrom, M. Cahoon, D. Belorgey, N. Srividya, H. Rubin, and J. G. Bieth
Comparative Trajectories of Active and S195A Inactive Trypsin upon Binding to Serpins
J. Biol. Chem., October 4, 2002; 277(41): 38901 - 38914.
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