C型肝炎は世界の人口の約３％にあたる１７０億人もの人々に感染するウイルス性の病気であり、肝臓における主たる病気となっている

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1 C 4 CD81 Crystal structure of the human CD81 extracellular domain, a four transmembrane receptor for Hepatitis C Virus. Kengo Kitadokoro 1,2, Domenico Bordo 1,3, Giuliano Galli 4, Roberto Petracca 4, Fabiana Falugi 4, Sergio Abrignani 4, Guido Grandi 4 and Martino Bolognesi 1 1Department of Physics - INFM and Advanced Biotechnology Center, University of Genoa, Via Dodecaneso, 33. I Genova (Italy). 2 Research Center for Instrumental Analysis, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto (Japan). 3 National Institute for Cancer Research c/o Advanced Biotechnology Center, Largo Rosanna Benzi,10. I Genova (Italy) 4 Chiron Vaccines Research Center, Via Fiorentina, Siena (Italy) Human CD81, which is belonged to tetraspanin family, has been previously identified as a receptor for the hepatitis C virus envelope E2 glycoprotein. The crystal structure of the human CD81 long extracellular domain, binding site for E2 glycoprotein, is presented here at 1.6 resolution. The tertiary structure of CD81-LEL, which is composed of five α-helices, is resemble for a mushroom-shaped molecules(stalk and head subdomains) and forms a dimer in the crystallographic asymmetric unit. The two disulfide bridges, which are conserved all the tetraspanin and are necessary for CD81-HCV interaction, are stabilizing the conformation of the head domain. This head domain is solvent exposed surface region and is locating the amino acid residues which is essential for the E2 binding. The hydrophobic cluster in this head domain may suggest that the presence of a docking site for a low complementary surface cavity in the partner E2 glycoprotein. We proposed that the dimer structure may be important in the interactions of HCV E2 glycoprotein

2 and also the viral protein may occur in dimeric aggregation on the HCV envelope. This common structural motifs of the tetraspanin provides the first insight onto the mechanism of HCV binding to human cell and may be targets for structure-based antiviral drug. C C HCV 9600 (+) RNA Choo 4 in vitro HCV SEL LEL Extracellular TM1 TM2 TM3 TM4 Cytoplasm N CY 1 CY2 CY3 C 1. 4 Tetraspanin

Chiron Pileri HCV E2 CD81 LEL Large Extracellular Loop 5 CD81 Tetraspanin family 6 CD81 236 SEL LEL CY1-3 TM1-TM4 CD81 LEL

3 Chiron Pileri HCV E2 CD81 LEL Large Extracellular Loop 5 CD81 Tetraspanin family 6 CD SEL LEL CY1-3 TM1-TM4 CD81 LEL Large Extracellular Loop Tetraspain HCV E2 55 Da CD81 LEL C 5 CD81 Tetraspanin family HCV E2 CD81 HCV HCV CD81 LEL X CD81 7

4 CD81 LEL GST ProteinA IgG 8 9 IgG ProteinA C Thrombin CD81-LEL His-tag IPTG L IgG IgG Ni P21 a = 31.5, b = 77.2, c = 38.5, Grenoble 1.6 ESRF 9 50 MAD SHARP/SOLOMON MAPMAN O Bones DM Averaging SHARP/SOLOMON 2 80 CNS REFMAC R = 18.7 R-free 23.8

5 CD81-LEL CD81-LEL 2 Large Extracellular loop 5 - A E A E "stalk" "head" "mushroom-like" 3, 4 7 CD81-LEL S-S Disorder 3 Molscript Raster3D Render

CD81-LEL N C stalk head DARI ESRF 100K S-S 10 1.

6 CD81-LEL N C stalk head DARI ESRF 100K S-S S-S S-S S-S HCV E S-S stalk head head C D A B 3 disorder

7 HCV Stalk A E Lys124 Asp195 Asp128 His CD81-LEL CD81-LEL N C TM3 TM4 head B C D CD81-LEL stalk 2 A A C Val114, Ile119, Val123, Phe126, Leu197, Phe198 CD81-LEL Tetraspanin Tetraspanin C D Ile181, Ile182, Leu185, Phe186 CD81

9 head HCV E2 CD81-LEL E2 12 CD81-LEL E2 HCV 5 AGM CD81-LEL E2 HCV 13 Tamarin TAM CD81-LEL E2 HCV 13 CD81-LEL AGM TAM 5 head 3 single-site mutation E2 Phe186 HCV-E2 12 CD81-LEL Phe186 Ala E2 AGM CD81-LEL Leu186 Phe E2 TAM Phe186 Phe186 Thr163 Ala Asp196 Glu E2 CD81 E2 Phe186 Phe186 head Phe186 HCV E2 Ile181, Ile182, Leu185 CD81-LEL E2

10 F 186. Dino CD81-LEL.

11 4 Tetraspanin Tetraspanin 14 6, 15 Tetraspanin CD81 CD9 CD9 16 CD9 CD9 17, 18 CD9 CD82 CD81 T human T lymphotropic virus 1, T 1 human T cell leukimia virus type 1, HTLV 1 CD82 19 Tetraspanin CD81 LEL Tetraspanin stalk C Tetraspanin Tetraspanin Tetraspanin Tetraspanin 7 CD81 CD9 Molecular Partner 20 CD81 21, 22 head Tetraspanin Tetraspan-Web 7, 23 HCV HTLV-1

12 Web LEL C CD81 LEL C HCV Tetraspanin Grenoble ESRF ID14 Hamburg EMBL/DESY 1. World Health Organization, (1997) Weekly Epidemiol. Rec. 72, Hoofnagle, J. H. (1997) Hepatology 26 (suppl. 1), 15S. 3. Houghton, M. (1996) in Virology, Fields, B. N., Knipe, D. M. and Howley, P. M. Eds. (Lippincott-Raven, Philadelphia, PA), Choo, Q. L., et al., (1989) Isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome. Science 244, Pileri, P., et al. (1998) Binding of hepatitis C virus to CD81. Science, 282, Levy, S., Todd, S. C. and Maecker, H. T. (1998) CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system. Annu. Rev. Immunol.. 16, Kitadokoro K., et al. (2001) CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs. EMBO J., 20, Nilsson, B., et al. (1987) A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng., 1, Kitadokoro, K., et al. (2001) Crystallization and preliminary crystallographic

13 studies on the large extracellular domain of human CD81, a tetraspanin receptor for Hepatitis C Virus. Acta Crystallogr. D57, Ravelli, R. B., McSweeney, S. M. (2000) The 'fingerprint' that X-rays can leave on structures. Structure Fold Des, 8, Petracca, R., et al. (2000) Structure-function analysis of hepatitis C virus envelope-cd81 binding. J. Virol., 74, Higginbottom, A., et al. (2000) Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2. J. Virol., 74, Meola, A., et al. (2000) Binding of hepatitis C virus E2 glycoprotein to CD81 does not correlate with species permissiveness to infection. J.Virol., 74, Hotta, H., et al (1988) Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Res. 48, Maecker, H. T., Todd, S. C. and Levy, S. (1997) The tetraspanin superfamily: molecular facilitators. FASEB J., 11, Iwamoto R., et al (1994) Heparin-binding EGF-like growth factor, which acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9, which up-regulates functional receptors and diphtheria toxin sensitivity. EMBO J., 13, Miyado K., et al (2000) Requirement of CD9 on the egg plasma membrane for fertilization. Science, 287, Le Naour F, et al (2000) Severely reduced female fertility in CD9-deficient mice. Science, 287, Pique C, et al (2000) Interaction of CD82 tetraspanin proteins with HTLV-1 envelope glycoproteins inhibits cell-to-cell fusion and virus transmission. Virology, 276, Charrin S, et al. (2001) The major CD9 and CD81 molecular partner. Identification and characterization of the complexes. J. Biol. Chem., 276, Hemler, M. E. (1998) Integrin associated proteins. Curr. Opin. Cell. Biol., 10, Serru, V., et al. (1999) Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions. Biochem. J., 340, Rubinstein E., et al. (1996) CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins. Eur. J. Immunol., 26,

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