Journal of Biochemistry Advance Access originally published online on January 3, 2007
Journal of Biochemistry 2007 141(2):269-277; doi:10.1093/jb/mvm037
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© 2007 The Japanese Biochemical Society.
RNA Recognition Mechanism of the Minimal Active Domain of the Human Immunodeficiency Virus Type-2 Nucleocapsid Protein
1Department of Physics, School of Science, and 2Department of Immunology, School of Allied Health Sciences, Kitasato University, Sagamihara, Kanagawa 228-8555; 3Department of Environmental Science, School of Human Environmental Science, Fukuoka Women's University, Higashi-ku, Fukuoka 813-8529; and 4Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo 194-8511, Japan
*To whom correspondence should be addressed. Tel: +81-42-778-9540, Fax: +81-42-778-9541, E-mail: kodera{at}kitasato-u.ac.jp
Received October 14, 2006; Accepted December 10, 2006
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Abstract |
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NCp8 of HIV-2 contains two CCHC-type zinc fingers connected by a linker, and is involved in many critical steps of the virus life cycle. It was previously shown that the first zinc finger flanked by the linker is the minimal active domain for specific binding to viral RNA. In our previous study, we determined the three-dimensional structure of NCp8-f1, including the minimal active domain, and found that a hydrogen bond between Asn11 N
H and Arg27 O stabilized the conformation of the linker in the vicinity of the zinc finger [Kodera et al. (1998) Biochemistry 37, 17704–17713]. In this study, RNA binding activities of NCp8-f1 and three types of its mutant peptides were analysed by native PAGE assay. The activity and three-dimensional structure of NCp8-f1/N11A, in which alanine is substituted for Asn11 thereby affecting the conformation of the linker, was analyzed and compared with those of NCp8-f1. We demonstrated that the existence of Arg4 and/or Lys5 and Arg26 and/or Arg27 were necessary for binding RNA. Furthermore, the linker's flexible orientation, which is controlled by the hydrogen bond between Asn11 NH and Arg27 O, appears to be a structural basis for NCp8 existing as a multi-functional protein.
Key Words: HIV, NMR, nucleocapsid protein, RNA binding protein, zinc finger
Abbreviations:
CCHC, Cys-X2-Cys-X4-His-X4-Cys; DQF-COSY, double quantum filtered-correlation spectroscopy; DSS, 4,4-dimethyl-4-silapentane-1-sulphonic acid; HIV, human immunodeficiency virus; HIV-1, HIV type-1; HIV-2, HIV type-2; HPLC, high performance liquid chromatography; MoMuLV, moloney murine leukemia virus; NC, nucleocapsid; NCp7, HIV-1 NC protein; NCp8, HIV-2 NC protein; NCp10, MoMuLV NC protein; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; RMSD, root-mean-squared deviation; SL, stem loop; TOCSY, total correlation spectroscopy; 
site, packaging signal site; WATERGATE, water suppression by gradient-tailored excitation