Mechanistic Role of an NS4A Peptide Cofactor with the Truncated NS3 Protease of Hepatitis C Virus:  Elucidation of the NS4A Stimulatory Effect via Kinetic Analysis and Inhibitor Mapping

Abstract

Infection by hepatitis C viruses (HCVs) is a serious medical problem with no broadly effective treatment available for the progression of chronic hepatitis. The catalytic activity of a viral serine protease located in the N-terminal one-third of nonstructural protein 3 (NS3) is required for polyprotein processing at four site-specific junctions. The three-dimensional crystal structure of the NS3-NS4A co-complex [Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R., & Thomson, J. A. (1996) Cell 87, 343-355] delineates a small hydrophobic region within the 54-residue NS4A protein that intercalates with and makes extensive contacts to the core of the protease. The current investigation addresses the mechanism of NS3 protease catalytic activation by NS4A utilizing a small synthetic NS4A peptide (residues 1678-1691 of the virus polyprotein sequence) and the recombinantly expressed protease domain of NS3. The addition of NS4A dramatically increased NS3 kcat and kcat/Km catalytic parameters when measured against small peptide substrates representing the different site-specific junctions of the polyprotein. The catalytic effect of natural and non-natural amino acid substitutions at the P1 position in a 5A/5B peptide substrate was investigated. NS3-NS4A demonstrated a marked catalytic preference for the cysteine residue commonly found in authentic substrates. The pH dependence of the NS3 hydrolysis reaction is not affected by the presence of NS4A. This result suggests that NS4A does not change the pKa values of the active site residues of NS3 protease. A steady state kinetic analysis was performed and indicated that the binding of NS4A and the peptide substrate occurs in an ordered fashion during the catalytic cycle, with NS4A binding first. Two distinct kinetic classes of peptidyl inhibitors based upon the 5A/5B cleavage site were identified. An NS4A-independent class is devoid of prime residues. A second class of inhibitors is NS4A-dependent and contains a natural or non-natural cyclic amino acid substituted for the commonly found P1' residue serine. These inhibitors display an up to 80-fold increase in affinity for NS3 protease in the presence of NS4A. Sequential truncation of prime and P residues from this inhibitor class demonstrated the fact that the P4' and P1' residues are crucial for potent inhibition. The selectivity of this NS4A effect is interpreted using a model of the 5A/5B decapeptide substrate bound to the active site of the NS3-NS4A structure.

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

“…Our results in solution thus are in contrast with the conclusions drawn by Love et al (1998) based on the crystallographic structures alone. On the other hand, our results are in agreement with the biochemical evidence that the presence of NS4A is not affecting the pK a of the catalytic residues (Landro et al, 1997), indicating that the catalytic triads of the enzyme-substrate complex and of the ternary enzyme-substrate-NS4A complex must possess very similar geometries.…”

“…These conclusions ®nd experimental support from a steady-state kinetic analysis of inhibitor binding to the active site of the NS3 proteinase (Landro et al, 1997). In fact, two classes of competitive inhibitors could be identi®ed: those interacting Root-mean-square deviation (A Ê ) comparison of the SRC backbone residues heavy atoms for several serine proteinases.…”

“…In fact, the pHdependence of the hydrolysis reaction studied in the presence of substrate and with or without NS4A titrates with the identical pK value of 7.2 (Landro et al, 1997). The authors conclude that the activation role of NS4A is not exerted by a perturbation of the pK a values of the active-site residues involved in the catalysis, in contrast with the model proposed by Love et al (1998).…”

“…On the other hand, NMR pH titration of the catalytic H57 residue of the free enzyme gives a pK a value of 6.8 . Assuming that the pK measured by Landro et al (1997) re¯ects the actual value for H57, the difference in pK a value from that of the free enzyme could be due either to a direct role of the substrate itself in the pK a alteration or to the different buffers used in the experiments. A kinetic analysis conducted on different types of substrate-like inhibitors in the absence and presence of the NS4A cofactor has shown that the action of NS4A peptide is exerted only on the P H -side of the substrate (Landro et al, 1997).…”

Prime Site Binding Inhibitors of a Serine Protease: NS3/4A of Hepatitis C Virus

“…In this respect, they were thus similar to previously reported examples of serine protease inhibitors spanning both enzyme subsites (4,(24)(25)(26)(27)(28)(29)(30)(31). Here we report on further work, which led to the development of peptidic and nonpeptidic inhibitors that bind exclusiVely to the prime site of NS3/4A.…”