Abstract
HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using 13C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome.
Highlights
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a small (55 amino acids) basic protein characterized by two zinc fingers and a basic N-terminal domain [1,2]
We showed that the binding is almost restricted to a single site: the TGG sequence located at the 39-end of the lower stem which alternates between single-stranded and double-stranded states [25], as a consequence of the destabilization induced by the internal loop
Addition of increasing amounts of NC(11–55) to native mini-cTAR resulted in the appearance of band CI, consistent with the 1:1 NC(11–55):mini-cTAR complex evidenced by NMR (Figure 2A)
Summary
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a small (55 amino acids) basic protein characterized by two zinc fingers and a basic N-terminal domain [1,2]. NC can bind non to any DNA and RNA sequence of 5–7 nt length. High-resolution structures allow understanding the structural basis for this specificity, by showing that insertion of an unpaired guanine into the hydrophobic platform at the top of the folded zinc fingers is systematically present in all solved complexes. This insertion is thought to be critical for discriminating the guanine residue from the other bases [9,11,19,20,21]
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