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. 1997 Mar;71(3):2436–2448. doi: 10.1128/jvi.71.3.2436-2448.1997

The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition.

E M Bergmann 1, S C Mosimann 1, M M Chernaia 1, B A Malcolm 1, M N James 1
PMCID: PMC191354  PMID: 9032381

Abstract

The virally encoded 3C proteinases of picornaviruses process the polyprotein produced by the translation of polycistronic viral mRNA. The X-ray crystallographic structure of a catalytically active mutant of the hepatitis A virus (HAV) 3C proteinase (C24S) has been determined. Crystals of this mutant of HAV 3C are triclinic with unit cell dimensions a = 53.6 A, b = 53.5 A, c = 53.2 A, alpha = 99.1 degrees, beta = 129.0 degrees, and gamma = 103.3 degrees. There are two molecules of HAV 3C in the unit cell of this crystal form. The structure has been refined to an R factor of 0.211 (Rfree = 0.265) at 2.0-A resolution. Both molecules fold into the characteristic two-domain structure of the chymotrypsin-like serine proteinases. The active-site and substrate-binding regions are located in a surface groove between the two beta-barrel domains. The catalytic Cys 172 S(gamma) and His 44 N(epsilon2) are separated by 3.9 A; the oxyanion hole adopts the same conformation as that seen in the serine proteinases. The side chain of Asp 84, the residue expected to form the third member of the catalytic triad, is pointed away from the side chain of His 44 and is locked in an ion pair interaction with the epsilon-amino group of Lys 202. A water molecule is hydrogen bonded to His 44 N(delta1). The side-chain phenolic hydroxyl group of Tyr 143 is close to this water and to His 44 N(delta1) and may be negatively charged. The glutamine specificity for P1 residues of substrate cleavage sites is attributed to the presence of a highly conserved His 191 in the S1 pocket. A very unusual environment of two water molecules and a buried glutamate contribute to the imidazole tautomer believed to be important in the P1 specificity. HAV 3C proteinase has the conserved RNA recognition sequence KFRDI located in the interdomain connection loop on the side of the molecule diametrically opposite the proteolytic site. This segment of polypeptide is located between the N- and C-terminal helices, and its conformation results in the formation of a well-defined surface with a strongly charged electrostatic potential. Presumably, this surface of HAV 3C participates in the recognition of the 5' and 3' nontranslated regions of the RNA genome during viral replication.

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Selected References

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  1. Abul Qasim M., Ranjbar M. R., Wynn R., Anderson S., Laskowski M., Jr Ionizable P1 residues in serine proteinase inhibitors undergo large pK shifts on complex formation. J Biol Chem. 1995 Nov 17;270(46):27419–27422. doi: 10.1074/jbc.270.46.27419. [DOI] [PubMed] [Google Scholar]
  2. Allaire M., Chernaia M. M., Malcolm B. A., James M. N. Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases. Nature. 1994 May 5;369(6475):72–76. doi: 10.1038/369072a0. [DOI] [PubMed] [Google Scholar]
  3. Andino R., Rieckhof G. E., Achacoso P. L., Baltimore D. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA. EMBO J. 1993 Sep;12(9):3587–3598. doi: 10.1002/j.1460-2075.1993.tb06032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bazan J. F., Fletterick R. J. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7872–7876. doi: 10.1073/pnas.85.21.7872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bode W., Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem. 1992 Mar 1;204(2):433–451. doi: 10.1111/j.1432-1033.1992.tb16654.x. [DOI] [PubMed] [Google Scholar]
  6. Burley S. K., Petsko G. A. Amino-aromatic interactions in proteins. FEBS Lett. 1986 Jul 28;203(2):139–143. doi: 10.1016/0014-5793(86)80730-x. [DOI] [PubMed] [Google Scholar]
  7. Chothia C. Principles that determine the structure of proteins. Annu Rev Biochem. 1984;53:537–572. doi: 10.1146/annurev.bi.53.070184.002541. [DOI] [PubMed] [Google Scholar]
  8. Cohen J. I., Ticehurst J. R., Purcell R. H., Buckler-White A., Baroudy B. M. Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses. J Virol. 1987 Jan;61(1):50–59. doi: 10.1128/jvi.61.1.50-59.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dougherty W. G., Semler B. L. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol Rev. 1993 Dec;57(4):781–822. doi: 10.1128/mr.57.4.781-822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fujinaga M., Delbaere L. T., Brayer G. D., James M. N. Refined structure of alpha-lytic protease at 1.7 A resolution. Analysis of hydrogen bonding and solvent structure. J Mol Biol. 1985 Aug 5;184(3):479–502. doi: 10.1016/0022-2836(85)90296-7. [DOI] [PubMed] [Google Scholar]
  11. Fujinaga M., Sielecki A. R., Read R. J., Ardelt W., Laskowski M., Jr, James M. N. Crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution. J Mol Biol. 1987 May 20;195(2):397–418. doi: 10.1016/0022-2836(87)90659-0. [DOI] [PubMed] [Google Scholar]
  12. Gauss-Müller V., Jürgensen D., Deutzmann R. Autoproteolytic cleavage of recombinant 3C proteinase of hepatitis A virus. Virology. 1991 Jun;182(2):861–864. doi: 10.1016/0042-6822(91)90630-t. [DOI] [PubMed] [Google Scholar]
  13. Gorbalenya A. E., Donchenko A. P., Blinov V. M., Koonin E. V. Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold. FEBS Lett. 1989 Jan 30;243(2):103–114. doi: 10.1016/0014-5793(89)80109-7. [DOI] [PubMed] [Google Scholar]
  14. Gorbalenya A. E., Donchenko A. P., Blinov V. M., Koonin E. V. Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold. FEBS Lett. 1989 Jan 30;243(2):103–114. doi: 10.1016/0014-5793(89)80109-7. [DOI] [PubMed] [Google Scholar]
  15. Hanecak R., Semler B. L., Ariga H., Anderson C. W., Wimmer E. Expression of a cloned gene segment of poliovirus in E. coli: evidence for autocatalytic production of the viral proteinase. Cell. 1984 Jul;37(3):1063–1073. doi: 10.1016/0092-8674(84)90441-0. [DOI] [PubMed] [Google Scholar]
  16. Harmon S. A., Updike W., Jia X. Y., Summers D. F., Ehrenfeld E. Polyprotein processing in cis and in trans by hepatitis A virus 3C protease cloned and expressed in Escherichia coli. J Virol. 1992 Sep;66(9):5242–5247. doi: 10.1128/jvi.66.9.5242-5247.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harris K. S., Xiang W., Alexander L., Lane W. S., Paul A. V., Wimmer E. Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding. J Biol Chem. 1994 Oct 28;269(43):27004–27014. [PubMed] [Google Scholar]
  18. Heinz B. A., Tang J., Labus J. M., Chadwell F. W., Kaldor S. W., Hammond M. Simple in vitro translation assay to analyze inhibitors of rhinovirus proteases. Antimicrob Agents Chemother. 1996 Jan;40(1):267–270. doi: 10.1128/aac.40.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hutchinson E. G., Thornton J. M. PROMOTIF--a program to identify and analyze structural motifs in proteins. Protein Sci. 1996 Feb;5(2):212–220. doi: 10.1002/pro.5560050204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hämmerle T., Molla A., Wimmer E. Mutational analysis of the proposed FG loop of poliovirus proteinase 3C identifies amino acids that are necessary for 3CD cleavage and might be determinants of a function distinct from proteolytic activity. J Virol. 1992 Oct;66(10):6028–6034. doi: 10.1128/jvi.66.10.6028-6034.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ivanoff L. A., Towatari T., Ray J., Korant B. D., Petteway S. R., Jr Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5392–5396. doi: 10.1073/pnas.83.15.5392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. James M. N., Sielecki A. R., Brayer G. D., Delbaere L. T., Bauer C. A. Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8 A resolution. A model for serine protease catalysis. J Mol Biol. 1980 Nov 25;144(1):43–88. doi: 10.1016/0022-2836(80)90214-4. [DOI] [PubMed] [Google Scholar]
  23. Jewell D. A., Swietnicki W., Dunn B. M., Malcolm B. A. Hepatitis A virus 3C proteinase substrate specificity. Biochemistry. 1992 Sep 1;31(34):7862–7869. doi: 10.1021/bi00149a017. [DOI] [PubMed] [Google Scholar]
  24. Jia X. Y., Summers D. F., Ehrenfeld E. Primary cleavage of the HAV capsid protein precursor in the middle of the proposed 2A coding region. Virology. 1993 Mar;193(1):515–519. doi: 10.1006/viro.1993.1157. [DOI] [PubMed] [Google Scholar]
  25. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  26. Kräusslich H. G., Wimmer E. Viral proteinases. Annu Rev Biochem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
  27. Leong L. E., Walker P. A., Porter A. G. Human rhinovirus-14 protease 3C (3Cpro) binds specifically to the 5'-noncoding region of the viral RNA. Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities. J Biol Chem. 1993 Dec 5;268(34):25735–25739. [PubMed] [Google Scholar]
  28. Malcolm B. A., Chin S. M., Jewell D. A., Stratton-Thomas J. R., Thudium K. B., Ralston R., Rosenberg S. Expression and characterization of recombinant hepatitis A virus 3C proteinase. Biochemistry. 1992 Apr 7;31(13):3358–3363. doi: 10.1021/bi00128a008. [DOI] [PubMed] [Google Scholar]
  29. Malcolm B. A. The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds. Protein Sci. 1995 Aug;4(8):1439–1445. doi: 10.1002/pro.5560040801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Martin A., Escriou N., Chao S. F., Girard M., Lemon S. M., Wychowski C. Identification and site-directed mutagenesis of the primary (2A/2B) cleavage site of the hepatitis A virus polyprotein: functional impact on the infectivity of HAV RNA transcripts. Virology. 1995 Oct 20;213(1):213–222. doi: 10.1006/viro.1995.1561. [DOI] [PubMed] [Google Scholar]
  31. Matthews D. A., Smith W. W., Ferre R. A., Condon B., Budahazi G., Sisson W., Villafranca J. E., Janson C. A., McElroy H. E., Gribskov C. L. Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein. Cell. 1994 Jun 3;77(5):761–771. doi: 10.1016/0092-8674(94)90059-0. [DOI] [PubMed] [Google Scholar]
  32. Merritt E. A., Murphy M. E. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]
  33. Molla A., Harris K. S., Paul A. V., Shin S. H., Mugavero J., Wimmer E. Stimulation of poliovirus proteinase 3Cpro-related proteolysis by the genome-linked protein VPg and its precursor 3AB. J Biol Chem. 1994 Oct 28;269(43):27015–27020. [PubMed] [Google Scholar]
  34. Morris A. L., MacArthur M. W., Hutchinson E. G., Thornton J. M. Stereochemical quality of protein structure coordinates. Proteins. 1992 Apr;12(4):345–364. doi: 10.1002/prot.340120407. [DOI] [PubMed] [Google Scholar]
  35. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  36. Nienaber V. L., Breddam K., Birktoft J. J. A glutamic acid specific serine protease utilizes a novel histidine triad in substrate binding. Biochemistry. 1993 Nov 2;32(43):11469–11475. doi: 10.1021/bi00094a001. [DOI] [PubMed] [Google Scholar]
  37. Palmenberg A. C. Proteolytic processing of picornaviral polyprotein. Annu Rev Microbiol. 1990;44:603–623. doi: 10.1146/annurev.mi.44.100190.003131. [DOI] [PubMed] [Google Scholar]
  38. Palmenberg A. C., Rueckert R. R. Evidence for intramolecular self-cleavage of picornaviral replicase precursors. J Virol. 1982 Jan;41(1):244–249. doi: 10.1128/jvi.41.1.244-249.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Perona J. J., Craik C. S. Structural basis of substrate specificity in the serine proteases. Protein Sci. 1995 Mar;4(3):337–360. doi: 10.1002/pro.5560040301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Petithory J. R., Masiarz F. R., Kirsch J. F., Santi D. V., Malcolm B. A. A rapid method for determination of endoproteinase substrate specificity: specificity of the 3C proteinase from hepatitis A virus. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11510–11514. doi: 10.1073/pnas.88.24.11510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Read R. J., Fujinaga M., Sielecki A. R., James M. N. Structure of the complex of Streptomyces griseus protease B and the third domain of the turkey ovomucoid inhibitor at 1.8-A resolution. Biochemistry. 1983 Sep 13;22(19):4420–4433. doi: 10.1021/bi00288a012. [DOI] [PubMed] [Google Scholar]
  42. Rossmann M. G. The molecular replacement method. Acta Crystallogr A. 1990 Feb 1;46(Pt 2):73–82. doi: 10.1107/s0108767389009815. [DOI] [PubMed] [Google Scholar]
  43. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  44. Schultheiss T., Kusov Y. Y., Gauss-Müller V. Proteinase 3C of hepatitis A virus (HAV) cleaves the HAV polyprotein P2-P3 at all sites including VP1/2A and 2A/2B. Virology. 1994 Jan;198(1):275–281. doi: 10.1006/viro.1994.1030. [DOI] [PubMed] [Google Scholar]
  45. Schultheiss T., Sommergruber W., Kusov Y., Gauss-Müller V. Cleavage specificity of purified recombinant hepatitis A virus 3C proteinase on natural substrates. J Virol. 1995 Mar;69(3):1727–1733. doi: 10.1128/jvi.69.3.1727-1733.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Storer A. C., Ménard R. Catalytic mechanism in papain family of cysteine peptidases. Methods Enzymol. 1994;244:486–500. doi: 10.1016/0076-6879(94)44035-2. [DOI] [PubMed] [Google Scholar]
  47. Tesar M., Pak I., Jia X. Y., Richards O. C., Summers D. F., Ehrenfeld E. Expression of hepatitis A virus precursor protein P3 in vivo and in vitro: polyprotein processing of the 3CD cleavage site. Virology. 1994 Feb;198(2):524–533. doi: 10.1006/viro.1994.1063. [DOI] [PubMed] [Google Scholar]
  48. Wilmot C. M., Thornton J. M. Beta-turns and their distortions: a proposed new nomenclature. Protein Eng. 1990 May;3(6):479–493. doi: 10.1093/protein/3.6.479. [DOI] [PubMed] [Google Scholar]
  49. Xiang W., Harris K. S., Alexander L., Wimmer E. Interaction between the 5'-terminal cloverleaf and 3AB/3CDpro of poliovirus is essential for RNA replication. J Virol. 1995 Jun;69(6):3658–3667. doi: 10.1128/jvi.69.6.3658-3667.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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