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 hepatitis C virus Bk strain (NS3(1-180)) under different physico-chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS3(1-180) either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS3(1-180) in solution was essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher glycerol 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 interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N-terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N-terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.
Orrù
S
S
Centro Internazionale di Servizi di Spettrometria di Massa, CNR-Università di Napoli Federico II, Italy.
Dal Piaz
F
F
Casbarra
A
A
Biasiol
G
G
De Francesco
R
R
Steinkühler
C
C
Pucci
P
P
D000595:Amino Acid Sequence; D005990:Glycerol / Q000737:chemistry; D016174:Hepacivirus / Q000201:enzymology*; D006868:Hydrolysis; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D005990:Glycerol; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases