Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry

Conformational changes occurring within the NS3 protease domain from the he patitis C virus Bk strain (NS3(1-180)) under different physico-chemical con ditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface acces sibility of the protein is affected by conformational changes, when compara tive experiments were carried out on NS3(1-180) either at different glycero l concentrations or in the presence of Pep4A, differential peptide maps wer e obtained from which protein regions involved in the structural changes co uld be inferred. The surface topology of isolated NS3(1-180) in solution wa s essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher gl ycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N-terminal segment that either was forced to interact with the protein or originate intermolecular interaction s with neighboring molecules. Binding of the cofactor Pep4A caused the disp lacement of the N-terminal arm from the protein moiety, leading this segmen t to again adopt an open and flexible conformation, thus suggesting that th e N-terminus of the protease contributes only marginally to the stability o f the complex. The observed conformational changes might be directly correl ated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substra te binding site.