Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

Ines Benzaghou, Isabelle Bougie, Martin Bisaillon
2004 Journal of Biological Chemistry  
The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding
more » ... creases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp 220 , Asp 318 , and Asp 319 for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzymebound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme. Recent estimates indicate that more than 170 million people worldwide are infected with the hepatitis C virus (HCV) 1 (1). It is estimated that about 80% of patients with acute HCV infection will progress to chronic hepatitis. Of these, 20% will develop cirrhosis, and 1-5% will develop hepatocellular carcinoma (2-5). There is thus an urgent need for the development of antiviral drugs aimed at inhibiting this pathogen. The HCV nonstructural 5B protein (NS5B) has been shown to be an RNA-dependent RNA polymerase (6 -11). The protein contains characteristic motifs, such as the GDD motif, shared by RNA-dependent RNA polymerases, and is believed to be responsible for the genome replication of HCV (12). The NS5B protein has been studied extensively during the past few years because it is one of the major targets for the development of antiviral drugs (7, 13-25). The enzyme can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (26). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (3) . This lack of specificity for HCV RNA supports the notion that additional viral or cellular factors are required for specific recognition of the viral replication signal. The HCV RNA polymerase activity has been shown to be supported by both magnesium and manganese ions (7, 13, 21-25). However, the recent characterization of the affinity of the enzyme for metal ions suggests that magnesium is the cation that is used in vivo during polymerization (27). Analysis of the crystal structure of NS5B revealed that the protein is folded into characteristic fingers, palm, and thumb subdomains (28, 29) . The particular fold adopted by the palm subdomain is shared by many proteins that bind nucleotides and/or nucleic acids (30). The crystal structure of the HCV RNA polymerase showed that it contains two absolutely conserved aspartic acid residues that coordinate two metal ions in the active site of the protein (31). These two metal ions are in contact with both the phosphate of the nucleotide and several acidic amino acids residues (31). A catalytic mechanism has been proposed for polymerases in which one metal ion is involved in both positioning the substrate and in the activation of an incoming nucleophile (32). Nucleophilic attack would then generate a trigonal bipyramidal transition state that would be stabilized by both metal ions. The second metal ion also stabilizes the negative charge that appears on the leaving 3Ј-oxygen, thus facilitating its departure from the phosphate. Analysis of the NS5B protein crystal structure indicated that the two metal ions are about 3.6 Å apart in the active site of the protein (31). Crystallographic and fluorescence spectroscopy data indicate that metal ion binding seems to be limited to the active site region and does not involve other subdomains of the protein (27, 31). Finally, we and others recently demonstrated that the enzyme undergoes conformational changes upon binding of metal ions (27, 33). However, this process does not significantly stimulate the binding of the enzyme to the RNA or NTP substrates (27). In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the HCV RNA polymerase. Using fluorescence spectroscopy, circular dichroism (CD), and denaturation assays, we demonstrate that the binding of metal ions to the enzyme is critical for both structural stabilization and catalysis. Mutational analysis also
doi:10.1074/jbc.m409657200 pmid:15375162 fatcat:u5zhwansvvei7fxamgta5bnl5i