Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR.
The retroviral gag nucleocapsid protein NCp7 (72 amino acids) of HIV-1 (LAV strain), which contains two successive zinc fingers of the Cys-X2-Cys-X4-His-X4-Cys form linked by a stretch of basic residues, promotes viral RNA dimerization and encapsidation and activates annealing of the primer tRNA to the initiation site of reverse transcription. The structure of NCp7 and other shorter fragments was studied by 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution to account for its various biological properties. Complete sequence specific 1H NMR assignments of the 13-51 residues of NCp7 encompassing the two zinc fingers was achieved by two-dimensional NMR experiments and the three-dimensional structure of (13-51)NCp7 was deduced from DIANA calculations, using nuclear Overhauser effects as constraints. The structure of the zinc complexed form of NCp7 is characterized by a kink at the Pro31 level in the basic Arg29-Ala-Pro-Arg-Lys-Lys-Gly35 RNA binding linker leading to a proximity of the Lys14-Cys18 to the Gly35-Cys39 sequences, which belong to the folded proximal and distal zinc fingers, respectively. Accordingly, the aromatic residues Phe16 and Trp37 were found to be spatially close. The Lys33 and Lys34 side-chains involved in viral RNA dimerization were solvent exposed. The N- and C-terminal sequences of NCp7 behave as flexible independent domains. The proposed structure of NCp7 might be used to rationally design new anti-viral agents aimed at inhibiting its functions.
Morellet
N
N
Département de Chimie Organique, U 266 INSERM, UA 498 CNRS, UFR des Sciences Pharmaceutiques et Biologiques, Paris, France.
Jullian
N
N
De Rocquigny
H
H
Maigret
B
B
Darlix
J L
JL
Roques
B P
BP
D000595:Amino Acid Sequence; D001665:Binding Sites; D036022:Capsid Proteins; D003196:Computer Graphics; D015683:Gene Products, gag / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D014764:Viral Proteins; D015032:Zinc / Q000032:analysis / Q000378:metabolism; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D010455:Peptides; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D006859:Hydrogen; D015032:Zinc