Solution structure and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex

The backbone assignments, secondary structure, topology, and dynamics of th e single-chain hepatitis C virus NS3 protease NS4A cofactor complex have be en determined by NMR spectroscopy. Residues I34 to S181 of NS3 and the cent ral three residues of the NS4A cofactor were assigned and the secondary str ucture was verified for these residues. In several Xray structures of NS4A- bound NS3 protease, residues 1 to 28 are stabilized by crystal packing, whi ch allows for the formation of the A0 strand and alpha0 helix. In solution, these N-terminal residues are largely unassigned and no evidence of a well -structured A0 strand or alpha0 helix was detected. NOEs between residues i n the E1-F1 loop (containing D81) and the al helix (containing 1157) togeth er with the detection of a D81-H57 hydrogen bond indicate that in solution the catalytic triad (D81, H57, S139) of the protease is better ordered in t he presence of the NS4A cofactor. This is consistent with the earlier cryst allographic results and may explain the observed increase in catalytic acti vity of the enzyme due to NS4A binding. A model-free analysis of our relaxa tion data indicates substantial exchange rates for residues V51-D81, which comprise the upper part of the N-terminal beta -barrel. A comparison of che mical-shift differences between NS3 protease and the NS3 protease-NS4A comp lex shows extensive chemical-shift changes for residues V51-D81 indicating that non-local structural changes occur upon NS4A binding to the NS3 protea se that are propagated well beyond the protease-cofactor interaction site. This is consistent with crystallographic data that reveal large structural rearrangements of the strand and loop regions formed by residues V51-D81 as a result of NS4A binding. The coincidence of large exchange rates for the NS3 protease-NS4A complex with chemical-shift differences due to NS4A bindi ng suggests that residues V51-D81 of the NS3 protease NS4A complex are in s low exchange with a NS4A-free conformation of NS3 protease. (C) 2001 Academ ic Press.