Skip to main content
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 Apr;7(4):837–847. doi: 10.1002/pro.5560070402

Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form.

Y Yan 1, Y Li 1, S Munshi 1, V Sardana 1, J L Cole 1, M Sardana 1, C Steinkuehler 1, L Tomei 1, R De Francesco 1, L C Kuo 1, Z Chen 1
PMCID: PMC2143993  PMID: 9568891

Abstract

The crystal structure of the NS3 protease of the hepatitis C virus (BK strain) has been determined in the space group P6(3)22 to a resolution of 2.2 A. This protease is bound with a 14-mer peptide representing the central region of the NS4A protein. There are two molecules of the NS3(1-180)-NS4A(21'-34') complex per asymmetric unit. Each displays a familiar chymotrypsin-like fold that includes two beta-barrel domains and four short alpha-helices. The catalytic triad (Ser-139, His-57, and Asp-81) is located in the crevice between the beta-barrel domains. The NS4A peptide forms an almost completely enclosed peptide surface association with the protease. In contrast to the reported H strain complex of NS3 protease-NS4A peptide in a trigonal crystal form (Kim JL et al., 1996, Cell 87:343-355), the N-terminus of the NS3 protease is well-ordered in both molecules in the asymmetric unit of our hexagonal crystal form. The folding of the N-terminal region of the NS3 protease is due to the formation of a three-helix bundle as a result of crystal packing. When compared with the unbound structure (Love RA et al., 1996, Cell 87:331-342), the binding of the NS4A peptide leads to the ordering of the N-terminal 28 residues of the NS3 protease into a beta-strand and an alpha-helix and also causes local rearrangements important for a catalytically favorable conformation at the active site. Our analysis provides experimental support for the proposal that binding of an NS4A-mimicking peptide, which increases catalytic rates, is necessary but not sufficient for formation of a well-ordered, compact and, hence, highly active protease molecule.

Full Text

The Full Text of this article is available as a PDF (9.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Kinetic and structural analyses of hepatitis C virus polyprotein processing. J Virol. 1994 Aug;68(8):5045–5055. doi: 10.1128/jvi.68.8.5045-5055.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol. 1993 Jul;67(7):3835–3844. doi: 10.1128/jvi.67.7.3835-3844.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blevins R. A., Tulinsky A. The refinement and the structure of the dimer of alpha-chymotrypsin at 1.67-A resolution. J Biol Chem. 1985 Apr 10;260(7):4264–4275. doi: 10.2210/pdb5cha/pdb. [DOI] [PubMed] [Google Scholar]
  4. Chen P., Tsuge H., Almassy R. J., Gribskov C. L., Katoh S., Vanderpool D. L., Margosiak S. A., Pinko C., Matthews D. A., Kan C. C. Structure of the human cytomegalovirus protease catalytic domain reveals a novel serine protease fold and catalytic triad. Cell. 1996 Sep 6;86(5):835–843. doi: 10.1016/s0092-8674(00)80157-9. [DOI] [PubMed] [Google Scholar]
  5. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989 Apr 21;244(4902):359–362. doi: 10.1126/science.2523562. [DOI] [PubMed] [Google Scholar]
  6. De Francesco R., Urbani A., Nardi M. C., Tomei L., Steinkühler C., Tramontano A. A zinc binding site in viral serine proteinases. Biochemistry. 1996 Oct 15;35(41):13282–13287. doi: 10.1021/bi9616458. [DOI] [PubMed] [Google Scholar]
  7. Di Bisceglie A. M. Hepatitis C and hepatocellular carcinoma. Semin Liver Dis. 1995 Feb;15(1):64–69. doi: 10.1055/s-2007-1007263. [DOI] [PubMed] [Google Scholar]
  8. Eckart M. R., Selby M., Masiarz F., Lee C., Berger K., Crawford K., Kuo C., Kuo G., Houghton M., Choo Q. L. The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor. Biochem Biophys Res Commun. 1993 Apr 30;192(2):399–406. doi: 10.1006/bbrc.1993.1429. [DOI] [PubMed] [Google Scholar]
  9. Failla C., Tomei L., De Francesco R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol. 1994 Jun;68(6):3753–3760. doi: 10.1128/jvi.68.6.3753-3760.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993 Mar;67(3):1385–1395. doi: 10.1128/jvi.67.3.1385-1395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harrison S. C. Peptide-surface association: the case of PDZ and PTB domains. Cell. 1996 Aug 9;86(3):341–343. doi: 10.1016/s0092-8674(00)80105-1. [DOI] [PubMed] [Google Scholar]
  12. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Shimotohno K. Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5547–5551. doi: 10.1073/pnas.88.13.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hijikata M., Mizushima H., Akagi T., Mori S., Kakiuchi N., Kato N., Tanaka T., Kimura K., Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993 Aug;67(8):4665–4675. doi: 10.1128/jvi.67.8.4665-4675.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hijikata M., Mizushima H., Tanji Y., Komoda Y., Hirowatari Y., Akagi T., Kato N., Kimura K., Shimotohno K. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10773–10777. doi: 10.1073/pnas.90.22.10773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell. 1996 Oct 18;87(2):343–355. doi: 10.1016/s0092-8674(00)81351-3. [DOI] [PubMed] [Google Scholar]
  16. Kleywegt G. J., Brünger A. T. Checking your imagination: applications of the free R value. Structure. 1996 Aug 15;4(8):897–904. doi: 10.1016/s0969-2126(96)00097-4. [DOI] [PubMed] [Google Scholar]
  17. Liddington R. C., Yan Y., Moulai J., Sahli R., Benjamin T. L., Harrison S. C. Structure of simian virus 40 at 3.8-A resolution. Nature. 1991 Nov 28;354(6351):278–284. doi: 10.1038/354278a0. [DOI] [PubMed] [Google Scholar]
  18. Lin C., Prágai B. M., Grakoui A., Xu J., Rice C. M. Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics. J Virol. 1994 Dec;68(12):8147–8157. doi: 10.1128/jvi.68.12.8147-8157.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lin C., Thomson J. A., Rice C. M. A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro. J Virol. 1995 Jul;69(7):4373–4380. doi: 10.1128/jvi.69.7.4373-4380.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lovejoy B., Choe S., Cascio D., McRorie D. K., DeGrado W. F., Eisenberg D. Crystal structure of a synthetic triple-stranded alpha-helical bundle. Science. 1993 Feb 26;259(5099):1288–1293. doi: 10.1126/science.8446897. [DOI] [PubMed] [Google Scholar]
  21. Qiu X., Culp J. S., DiLella A. G., Hellmig B., Hoog S. S., Janson C. A., Smith W. W., Abdel-Meguid S. S. Unique fold and active site in cytomegalovirus protease. Nature. 1996 Sep 19;383(6597):275–279. doi: 10.1038/383275a0. [DOI] [PubMed] [Google Scholar]
  22. Saito I., Miyamura T., Ohbayashi A., Harada H., Katayama T., Kikuchi S., Watanabe Y., Koi S., Onji M., Ohta Y. Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6547–6549. doi: 10.1073/pnas.87.17.6547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shieh H. S., Kurumbail R. G., Stevens A. M., Stegeman R. A., Sturman E. J., Pak J. Y., Wittwer A. J., Palmier M. O., Wiegand R. C., Holwerda B. C. Three-dimensional structure of human cytomegalovirus protease. Nature. 1996 Sep 19;383(6597):279–282. doi: 10.1038/383279a0. [DOI] [PubMed] [Google Scholar]
  24. Shimizu Y., Yamaji K., Masuho Y., Yokota T., Inoue H., Sudo K., Satoh S., Shimotohno K. Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease. J Virol. 1996 Jan;70(1):127–132. doi: 10.1128/jvi.70.1.127-132.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Takamizawa A., Mori C., Fuke I., Manabe S., Murakami S., Fujita J., Onishi E., Andoh T., Yoshida I., Okayama H. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol. 1991 Mar;65(3):1105–1113. doi: 10.1128/jvi.65.3.1105-1113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tanji Y., Hijikata M., Satoh S., Kaneko T., Shimotohno K. Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing. J Virol. 1995 Mar;69(3):1575–1581. doi: 10.1128/jvi.69.3.1575-1581.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tomei L., Failla C., Santolini E., De Francesco R., La Monica N. NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol. 1993 Jul;67(7):4017–4026. doi: 10.1128/jvi.67.7.4017-4026.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tomei L., Failla C., Vitale R. L., Bianchi E., De Francesco R. A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease. J Gen Virol. 1996 May;77(Pt 5):1065–1070. doi: 10.1099/0022-1317-77-5-1065. [DOI] [PubMed] [Google Scholar]
  29. Tong L., Qian C., Massariol M. J., Bonneau P. R., Cordingley M. G., Lagacé L. A new serine-protease fold revealed by the crystal structure of human cytomegalovirus protease. Nature. 1996 Sep 19;383(6597):272–275. doi: 10.1038/383272a0. [DOI] [PubMed] [Google Scholar]
  30. Tong L., Wengler G., Rossmann M. G. Refined structure of Sindbis virus core protein and comparison with other chymotrypsin-like serine proteinase structures. J Mol Biol. 1993 Mar 5;230(1):228–247. doi: 10.1006/jmbi.1993.1139. [DOI] [PubMed] [Google Scholar]
  31. Yan Y., Stehle T., Liddington R. C., Zhao H., Harrison S. C. Structure determination of simian virus 40 and murine polyomavirus by a combination of 30-fold and 5-fold electron-density averaging. Structure. 1996 Feb 15;4(2):157–164. doi: 10.1016/s0969-2126(96)00019-6. [DOI] [PubMed] [Google Scholar]
  32. Yan Y., Winograd E., Viel A., Cronin T., Harrison S. C., Branton D. Crystal structure of the repetitive segments of spectrin. Science. 1993 Dec 24;262(5142):2027–2030. doi: 10.1126/science.8266097. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES