Serine and Cysteine Proteases Are Translocated to Similar Extents upon Formation of Covalent Complexes with Serpins: FLUORESCENCE PERTURBATION AND FLUORESCENCE RESONANCE ENERGY TRANSFER MAPPING OF THE PROTEASE BINDING SITE IN CrmA COMPLEXES WITH GRANZYME B AND CASPASE-1

Richard Swanson,Manikanahally P Raghavendra,Weiqing Zhang,Christopher Froelich,Peter G.W Gettins,Steven T Olson

doi:10.1074/jbc.m609546200

Richard Swanson, Manikanahally P Raghavendra + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m609546200

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Abstract

CrmA is a "cross-class" serpin family inhibitor of the proapoptotic serine protease, granzyme B, as well as cysteine proteases of the caspase family. To determine whether crmA inhibits these structurally diverse proteases by a common conformational trapping mechanism, we mapped the position of the protease in crmA complexes with granzyme B or caspase-1 by fluorescence perturbation and fluorescence resonance energy transfer (FRET) analyses of site-specific fluorophore-labeled crmAs. A reactive loop P6 NBD label underwent similar large fluorescence enhancements (>200%) either upon reactive loop cleavage by AspN protease or complex formation with granzyme B or caspase-1, consistent with the insertion of the cleaved reactive loop into sheet A in both types of crmA-protease complexes. NBD labels on the noninserting part of the reactive loop docking site for protease (P1' residue) or midway between the two ends of sheet A (helix F residue 101) showed no significant perturbations due to protease complexation. By contrast, labels at positions 68 and 261, lying at the end of sheet A most distal from the reactive loop, showed marked perturbations distinct from those induced by AspN cleavage and thus ascribable to granzyme B or caspase-1 proximity in the complexes. Substantial FRET between protease tryptophans and 5-dimethylaminonaphthalene-1-sulfonyl-labeled crmAs occurred in protease complexes with crmAs labeled at the 68 and 261 positions, but not the P1' position. These results suggest that granzyme B and caspase-1 are inhibited by crmA by a common mechanism involving full reactive loop insertion into sheet A and translocation of the protease to the distal end of the sheet as previously found for inhibition of other serine proteases by serpins.

Highlights

The serpins are an ever expanding superfamily of proteins that are found in all types of organisms and thought to have an early evolutionary origin [1, 2]
Direct excitation of the dansyl fluorophore in 68DANS and 261DANS crmAs showed blue-shifts of the dansyl emission spectra in crmA-caspase-1 complexes relative to the unreacted labeled crmAs but minimal intensity changes (Ͻ10%) due to proximity effects of the complexed protease. These results are in agreement with the fluorescence perturbation studies and support the conclusion that the protease is localized in similar positions at the distal end of sheet A in crmA complexes with both serine and cysteine proteases. These studies were undertaken to determine whether serpins inhibit cysteine proteases by the same translocation and deformation mechanism that has been demonstrated for inhibition of serine proteases [12, 13]
That there might be significant differences in the mechanism of inhibition of these two mechanistically distinct classes of protease has been suggested by the fact that serpin complexes with cysteine proteases are not SDS-stable and may not be stabilized as acyl-intermediate complexes as is the case with serpin complexes with serine proteases [5, 8, 10]

Summary

Introduction

The serpins are an ever expanding superfamily of proteins that are found in all types of organisms and thought to have an early evolutionary origin [1, 2]. Our findings show that the protease is translocated to similar positions at the distal end of sheet A in stable crmA complexes with both granzyme B and caspase-1 and support similar conformational change-dependent mechanisms for serpin inhibition of the two classes of protease.

Results

Conclusion