Abstract
Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide1. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success2. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication3,4. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold5,6, but the enzymatic mechanism of the NS2-3 protease remains unresolved7,8,9. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 Å resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.
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Acknowledgements
We would like to thank R. MacKinnon, S. Darst and H. Mueller for the use of X-ray diffractometers, related equipment and software. We appreciate access to beamlines X9A and X29 of the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory and acknowledge the assistance of the NSLS staff. We wish to thank T. Tellinghuisen for assistance with data collection. D. Jeruzalmi provided the program msf_similarity_to_pdb and LOOSENGRASP. We would like to thank S. Burley, S. Darst, L. Dustin, M. Evans, C. Jones, C. Murray, B. Lindenbach, G. Randall and T. Tellinghuisen for input on the manuscript, and K. Kuo and S. You for help with artwork. I.C.L. was supported by fellowships from the Swiss National Science Foundation, the Roche Research Foundation and the Swiss Society for Medical-Biological Grants. J.M. was supported as a Merck Fellow of the Life Sciences Research Foundation. Additional financial support for this work came from grants from the National Institutes of Health and the Greenberg Medical Research Institute (C.M.R.). Author Contributions I.C.L., J.M. and C.M.R. conceived the experiments. I.C.L. generated all reagents, materials, proteins and crystals with assistance from J.M. All data collection and processing was done by J.M. and I.C.L. Model building and refinement were performed by I.C.L. and J.M. Cross-linking and mammalian expression studies were carried out by I.C.L. and T.G.D. The manuscript was written by I.C.L., J.M. and C.M.R.
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The atomic coordinates for this structure have been deposited in the Protein Data Bank under accession code 2HD0. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Supplementary information
Supplementary Table 1
Summary of crystal parameters, data collection and refinement statistics. (PDF 86 kb)
Supplementary Figure 1
Asymmetric unit of the native NS2pro crystal. (PDF 534 kb)
Supplementary Figure 2
Electron density maps of the linker polypeptide, the active site, the cis-proline residue, and a detergent molecule. (PDF 705 kb)
Supplementary Figure 3
Sequence conservation at the solvent-accessible surface of NS2pro. (PDF 235 kb)
Supplementary Figure 4
Dimerization of purified NS2pro in solution with a chemical crosslinker. (PDF 101 kb)
Supplementary Figure Legends
This file contains legends to the Supplementary Figures. (DOC 26 kb)
Supplementary Data
Supplementary Data nature04975-s7.doc This file contains a detailed description of the methods, and additional references. (DOC 68 kb)
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Lorenz, I., Marcotrigiano, J., Dentzer, T. et al. Structure of the catalytic domain of the hepatitis C virus NS2-3 protease. Nature 442, 831–835 (2006). https://doi.org/10.1038/nature04975
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DOI: https://doi.org/10.1038/nature04975
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