Site-specific characterization of HIV-1 nucleocapsid protein binding to oligonucleotides with two binding sites.
The nucleocapsid protein (NC) of HIV-1 is a highly conserved protein essential for the virus life cycle that constitutes an attractive target for new antiviral agents. Most NC functions rely on its binding to the HIV-1 genomic RNA and its DNA copies that contain multiple and possibly interdependent binding sites. Therefore, a detailed understanding of NC binding requires a site-specific experimental approach. We have recently shown that 2-aminopurine (2Ap), a fluorescent adenine analogue, can site-selectively probe the binding of NC. Here, we introduced 2Ap at various positions of model single-stranded dodecanucleotides containing two TG motifs which constitute putative specific binding sites. Steady-state and time-resolved fluorescence experiments indicated that NC binding strongly increased the fluorescence quantum yield of 2AP by reducing the dynamic quenching of 2Ap by its close neighbors and slowing the picosecond to nanosecond conformational fluctuations of the oligonucleotides. The dodecanucleotides were found to bind two NC molecules at physiological salt concentrations, confirming the preferential binding of NC to TG motifs and an occluded binding site size for NC of five to six bases. Using the NC-induced changes in 2Ap fluorescence, we determined the microscopic affinity constants of the individual binding sites and showed that affinities can significantly differ from one site to another within the same dodecanucleotide, depending on the position of the TG dinucleotide and the nature of its close neighbors. Moreover, our data suggest that binding of NC even to close binding sites shows no strong cooperativity.
Avilov
Sergiy V
SV
Laboratoire Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France.
Godet
Julien
J
Piémont
Etienne
E
Mély
Yves
Y
D015075:2-Aminopurine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D015497:HIV-1 / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*; D011485:Protein Binding; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry*
C071884:NCP7 protein, Human immunodeficiency virus 1; D009841:Oligonucleotides; D054301:gag Gene Products, Human Immunodeficiency Virus; D015075:2-Aminopurine