Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population1. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase2,3, as well as a pivotal regulator of replication2,4 and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth5,6. NS5A is a large phosphoprotein (56–58 kDa) with an amphipathic α-helix at its amino terminus that promotes membrane association7,8,9. After this helix region, NS5A is organized into three domains10. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule10. Mutations disrupting either the membrane anchor7,8 or zinc binding10 of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-Å resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.
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We would like to acknowledge R. MacKinnon, S. Darst and H. Mueller for the use of X-ray diffractometers, related equipment and software. We appreciate access to beamline X9A at the National Synchrotron Light Source (NSLS) at the Brookhaven National Labs and acknowledge the assistance of NSLS staff. J.-W. Carroll provided vital assistance with data collection. D. Jeruzalmi provided the program msf_similarity_to_pdb. We wish to thank S. Darst, M. Evans, A. Gauthier, C. Jones, B. Lindenbach, I. Lorenz, T. von Hahn, M. Tellinghuisen and K. Tellinghuisen for critical reading of this manuscript. T.L.T. was supported in part by fellowships from the Charles H. Revson Foundation for Biomedical Research and the National Institutes of Health Ruth L. Kirschstein National Research Service Award, granted through the National Institute of Allergy and Infectious Disease. 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 T.L.T., J.M. and C.M.R. conceived these experiments. T.L.T. generated all reagents, materials, proteins and crystals used herein, with assistance from J.M. All data collection was carried out by J.M. and T.L.T. Data processing, model building and refinement were performed by T.L.T., with significant input and assistance from J.M. The manuscript was written by T.L.T. with comments and assistance from J.M. and C.M.R.
The coordinates for this structure have been deposited in the Protein Data Bank (PDB) under accession code 1ZH1. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Sequence map of NS5A domain I. Secondary structures are shown and numbered as in the structure. Amino acids are coloured as follows: red, cysteines contacting zinc; green, disulphide bonded cysteines; grey, proline subdomain connector; blue, residues lining the basic groove; pink, dimer interface residues; and orange, conserved surface patch. (JPG 54 kb)
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Tellinghuisen, T., Marcotrigiano, J. & Rice, C. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 435, 374–379 (2005). https://doi.org/10.1038/nature03580
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