Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study.
The critical functions of the HIV-1 nucleocapsid protein NCp7 in genomic RNA packaging and reverse transcription, essentially rely on interactions with nucleic acids. A significant progress in the knowledge of these interactions has been recently achieved with the NMR-derived structures of NCp7 derivatives in complex with two short sequences of the HIV-1 psi packaging signal, namely ACGCC and the stem-loop 3 (SL3) motif. To further identify the key nucleotides in the formation of both NCp7-d(ACGCC) and NCp7-SL3 complexes, we quantitatively analyzed by steady-state and time-resolved fluorescence, the interaction of NCp7 with d(ACGCC) and SL3 mutants where each nucleotide in interaction with the protein has been systematically substituted. Moreover, by using several NCp7 derivatives, we investigated the contributions of Phe16, Trp37, and Trp61, and the various NCp7 domains, in the binding process. The binding of NCp7 appeared essentially driven by the interaction of the zinc finger domain and notably Trp37 with a G residue, irrespective of its location in the oligonucleotide. The involvement of Trp37 in the binding process depended on its location in the C-terminal finger motif and the proper folding of this motif. Phe16 in the N-terminal finger motif also strongly contributed to the binding energy, while in contrast, Trp61 in the C-terminal domain only marginally interacted with the oligonucleotides. The stem-loop structure of SL3 stabilized the binding of NCp7 by about -7 kJ/mol (at 0.1 M NaCl) by favoring the electrostatic binding of both N- and C-terminal domains. Finally, we found that NCp7 bound to nucleic acid single-stranded regions with the following preference: X(i)()TGX(j)() > X(i)()GXGX(j)() approximately X(i)()TXGX(j)() > X(i)()GX(j)() >> X(i)()X(j)(), where X corresponds to either A or C. This implies that recognition of nucleic acids by NCp7 may be achieved by a limited number of sites, and hence, no strong affinities are required in order to get a selective binding.
Vuilleumier
C
C
Laboratoire de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR 7034 CNRS, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg, France.
Bombarda
E
E
Morellet
N
N
Gérard
D
D
Roques
B P
BP
Mély
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins; D003854:Deoxyribonucleotides / Q000378:metabolism; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins; D019065:Virus Assembly / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D003854:Deoxyribonucleotides; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D009841:Oligonucleotides; D012367:RNA, Viral; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D010649:Phenylalanine; D006147:Guanine; D014364:Tryptophan