RNA recognition mechanism of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein.
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 Asn(11) N(delta)H and Arg(27) 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 Asn(11) thereby affecting the conformation of the linker, was analyzed and compared with those of NCp8-f1. We demonstrated that the existence of Arg(4) and/or Lys(5) and Arg(26) and/or Arg(27) were necessary for binding RNA. Furthermore, the linker's flexible orientation, which is controlled by the hydrogen bond between Asn(11) N(delta)H and Arg(27) O, appears to be a structural basis for NCp8 existing as a multi-functional protein.
Matsui
Takashi
T
Department of Physics, School of Science, Kitasato University, Sagamihara, Kanagawa 228-8555, Japan.
Kodera
Yoshio
Y
Endoh
Hiroshi
H
Miyauchi
Emi
E
Komatsu
Hiroyoshi
H
Sato
Kazuki
K
Tanaka
Takeshi
T
Kohno
Toshiyuki
T
Maeda
Tadakazu
T
D036022:Capsid Proteins / Q000737:chemistry* / Q000378:metabolism*; D015498:HIV-2 / Q000737:chemistry*; D006801:Humans; D009154:Mutation; D010455:Peptides; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D016335:Zinc Fingers
D036022:Capsid Proteins; D010455:Peptides; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C101406:nucleocapsid protein 8, HIV-2