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

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 increases 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 Asp220, Asp318, and Asp319 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 enzyme-bound 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. 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 increases 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 Asp220, Asp318, and Asp319 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 enzyme-bound 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) 1The abbreviations used are: HCV, hepatitis C virus; ANS, 1-anilino-8-naphthalenesulfonate; GdmHCl, guanidinium hydrochloride; NS5B, nonstructural 5B protein. (1Sarbah S. Younossi Z. J. Clin. Gastroenterol. 2000; 30: 125-143Crossref PubMed Scopus (131) Google Scholar). 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 (2Choo Q.L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6292) Google Scholar, 3Saito I. Miyamura T. Ohbayashi A. Harada H. Katayama T. Kikuchi S. Watanabe Y. Koi S. Onji M. Ohta Y. Choo Q.L. Houghton M. Kuo G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549Crossref PubMed Scopus (1085) Google Scholar, 4Alter H.J. Blood. 1995; 85: 1681-1695Crossref PubMed Google Scholar, 5Houghton M. Fields B.N. Knipe D.M. Howley P.M. Virology. Lippincott-Raven Press, Philadelphia1996: 1035-1058Google Scholar). 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 (6Al R.H. Xie Y. Wang Y. Hagedorn C.H. Virus Res. 1998; 53: 141-149Crossref PubMed Scopus (57) Google Scholar, 7Behrens S.E. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar, 8De Francesco R. Behrens S.E. Tomei L. Altamura S. Jiricny J. Methods Enzymol. 1996; 275: 58-67Crossref PubMed Scopus (64) Google Scholar, 9Lohmann V. Korner F. Herian U. Bartenschlager R. J. Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar, 10Yuan Z.H. Kumar U. Thomas H.C. Wen Y.M. Monjardino J. Biochem. Biophys. Res. Commun. 1997; 232: 231-235Crossref PubMed Scopus (63) Google Scholar, 11Oh J.W. Sheu G.T. Lai M.M. J. Biol. Chem. 2000; 275: 17710-17717Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). 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 (12Miller R.H. Purcell R.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2057-2061Crossref PubMed Scopus (531) Google Scholar). 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 (7Behrens S.E. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar, 13Ferrari E. Wright-Minogue J. Fang J.W. Baroudy B.M. Lau J.Y. Hong Z. J. Virol. 1999; 73: 1649-1654Crossref PubMed Google Scholar, 14Lohmann V. Roos A. Korner F. Koch J.O. Bartenschlager R. J. Viral Hepatitis. 2000; 7: 167-174Crossref PubMed Scopus (66) Google Scholar, 15Cheney I.W. Naim S. Lai V.C.H. Dempsey S. Bellows D. Walker M.P. Shim J.H. Horscroft N. Hong Z. Zhong W. Virology. 2002; 297: 298-306Crossref PubMed Scopus (49) Google Scholar, 16Kao C.C. Yang X. Kline A. Wang Q.M. Barket D. Heinz B.A. J. Virol. 2000; 74: 11121-11128Crossref PubMed Scopus (115) Google Scholar, 17Oh J.W. Ito T. Lai M.M.C. J. 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PubMed Scopus Google Scholar). a that be by both metal ions. The metal ion also the that on the thus the of the NS5B protein that the metal ions are about in the active site of the protein S. Tomei L. De Francesco R. J. Virol. 2002; PubMed Scopus Google Scholar). and indicate that metal ion binding to be to the active site region and other of the protein I. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Tomei L. De Francesco R. J. Virol. 2002; PubMed Scopus Google Scholar). Finally, we and that the binding of metal ions I. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. C. S. C.C. J. Virol. 2002; PubMed Scopus Google Scholar). this significantly the binding of the to the RNA I. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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. and denaturation assays, we demonstrate that the binding of metal ions to the is critical for both structural and analysis also revealed the importance of residues for metal binding. on the role of metal ions in the RNA polymerase HCV NS5B and and of a of HCV NS5B protein the of the protein performed as previously I. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the NS5B by the L.R. 1989; PubMed Scopus Google Scholar). was a was by the the of The to to the NS5B protein was by the of a of and with a The binding be by is the is the of the is the of and is the of The of protein as by is to by is the of the between the at a of and the in the of is the at and is the protein the is in to it be that is to and be to The a analysis of of of the for binding was to the that the and the the of of NS5B was to the of guanidinium and for at The parameters of in the of of and of denaturant to of the as previously and In of was as previously (7Behrens S.E. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar). The reaction was performed in of with of and The at for by and The was by performed with a The in with of by the in at The to at a of The results as in The of protein at each was by the is the for the enzyme. of was by the of at a of The to of HCV RNA by performed at by increasing of The was at and the to The between and was used for The in the presence of to the Biochem. PubMed Scopus Google and are the in the presence of the is the and is the the an the was and the to a is the the between and the enzyme. and of the HCV RNA the role of metal ions in the RNA polymerase the was as previously I. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). analysis that the NS5B protein was the in the The of the protein was also by a The of the NS5B protein in at are shown in of the of the NS5B protein revealed an that is to that of the is to be at of protein been to of the residues the a of the of the protein. denaturation of NS5B with results in a of of the of the of NS5B revealed a of protein This be to of binding performed at a protein of with the that the binding was by the presence of an a study, we that the HCV RNA polymerase the binding of metal this significantly the binding of RNA I. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). metal ions the to play both and structural in protein we the effect of ions on the structural stability of the HCV RNA polymerase. The effect of metal ion binding on the NS5B structural stability was by denaturation assays performed at an of the the of the NS5B protein to the of residues to a more The protein to the in the between and the on are be at than The of denaturant to of the was at denaturation assays performed in the presence of of ions. The thermodynamic parameters and the are in I. of the NS5B was in the presence of as by the in the that was at The is that the stability of the is increased by in the presence of parameters by in a The chemical denaturation assays also in of to study the effect of denaturation on the activity of the protein. In the first the HCV RNA polymerase was with increasing of The RNA polymerase activity of the protein was by the protein with and a of The that the RNA polymerase activity of the was by at a of In the the NS5B protein was with The was with and for RNA polymerase this a of was to the RNA polymerase activity by indicate that the binding of metal ions to the HCV RNA polymerase the chemical with the structural stability that the active site region of the is more sensitive to chemical denaturant than other structural scaffolds. The of metal binding on the structural stability of the HCV RNA polymerase also by denaturation assays performed in both the presence and of and of the was by the in the of the protein denaturation of the NS5B protein revealed a of thermal of The of of ions in a of the to results demonstrate that the binding of ions to the NS5B protein significantly increases the structural stability of the enzyme. stability was also in of This was by the NS5B protein the protein with ions for at by on The protein for RNA polymerase activity at in the RNA polymerase The thermal are shown in The results indicate that of the HCV RNA polymerase with ions the thermal The of the protein with ions is to the to the that been with metal ions that of the denaturation assays, performed with increasing by thermal revealed of a be during the a of and the HCV RNA polymerase has previously been shown to with metal ions I. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Tomei L. De Francesco R. J. Virol. 2002; PubMed Scopus Google Scholar, L. C. S. C.C. J. Virol. 2002; PubMed Scopus Google Scholar). In the present study, a thermodynamic of the metal ion binding was to the and of the binding the thermodynamic parameters of the binding reaction by the of the for ions. of the for the interaction between and the HCV RNA polymerase that the interaction is with a of Furthermore, the binding reaction is clearly with the indicate that the reaction is at to at the binding of metal ions be at than This be to the denaturation of the protein that at and the of the active at the structural that metal ion binding, we the binding of structural to the enzyme. The exposure of the hydrophobic of the HCV RNA polymerase was by the binding of to the protein. is a of hydrophobic on with to hydrophobic results in an of of Google Scholar). revealed that the NS5B protein to ANS, hydrophobic at the of the protein a of the is the protein is in the presence of of ions increasing the in a of the as the protein with by the Overall, indicate that ion binding increases hydrophobic exposure on the HCV RNA polymerase surface. the that the binding of ions to the HCV RNA performed in the presence of is a of the of the protein Biochemistry. PubMed Scopus Google Scholar). In the of for the NS5B protein to the structural between the NS5B protein and the protein to ions in our binding assays, has increased to the of the NS5B protein the protein is to ions The are in of the HCV RNA polymerase by in a The performed in the presence of increasing in metal ion binding the protein was with of This was in the presence of the binding of ions clearly the NS5B protein the assays performed in the presence of increasing indicate that ion binding to the HCV RNA polymerase significantly increases the structural stability of the protein. of the of the HCV RNA polymerase has revealed the presence of metal ions that in the active center of the S. Tomei L. De Francesco R. J. Virol. 2002; PubMed Scopus Google Scholar). The of and been shown to the metal the of Asp319 with one of the metal ions S. Tomei L. De Francesco R. J. Virol. 2002; PubMed Scopus Google Scholar). The importance of each of for catalysis has been previously by mutational analysis V. Korner F. Herian U. 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Finally, of the HCV RNA polymerase activity has also been interaction with the HCV and the of in to be The present has major in the structural stability of the HCV RNA polymerase it to ions. denaturation studies, coupled with polymerization assays, that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. mutational shown that the active site of HCV RNA polymerase is of an of and of a is for reaction V. Korner F. Herian U. Bartenschlager R. J. Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar). The that are the metal ions and in the NS5B active site also been by S. Tomei L. De Francesco R. J. Virol. 2002; PubMed Scopus Google Scholar). The of and the metal ions that in the the of Asp319 with one of the metal ions S. Tomei L. De Francesco R. J. Virol. 2002; PubMed Scopus Google Scholar). The importance of for catalysis has been by mutational to the residues the of the Asp319 by is V. Korner F. Herian U. Bartenschlager R. J. Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar). In to with the metal ions are in a of with the of S. Tomei L. De Francesco R. J. Virol. 2002; PubMed Scopus Google Scholar). metal is by of the metal with the in to the of the of the RNA during The present mutational analysis revealed the importance of each of the residues Asp318, and that the metal ions in the previously of the HCV RNA polymerase. The mutational study underscores a strict requirement for the by both and Asp318, the is that of the Asp319 by the binding The of the Asp319 clearly the geometry of the active center and the binding This that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, highlighting the critical role of the enzyme-bound metal ions. on the results of our and denaturation we that the NS5B protein a the binding of metal ions is by an increased stability of the enzyme. Furthermore, that the binding of metal ions significantly the binding of the HCV RNA polymerase to RNA I. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We thus that the is a for activity by the of residues in the active site of the at the to the of the we that ions play a dual modulatory role in the HCV RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme. the of the HCV replication and the and for is of the of NS5B the for in this Structural and are clearly to the of the polymerase to the of antiviral drugs to replication infected We for and with