Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine

Abstract

The nucleocapsid protein (NC) plays an important role in HIV-1, mainly through interactions with the genomic RNA and its DNA copies. Though the structures of several complexes of NC with oligonucleotides (ODNs) are known, detailed information on the ODN dynamics in the complexes is missing. To address this, we investigated the steady state and time-resolved fluorescence properties of 2-aminopurine (2Ap), a fluorescent adenine analog introduced at positions 2 and 5 of AACGCC and AATGCC sequences. In the absence of NC, 2Ap fluorescence was strongly quenched in the flexible ODNs, mainly through picosecond to nanosecond dynamic quenching by its neighboring bases. NC strongly restricted the ODN flexibility and 2Ap local mobility, impeding the collisions of 2Ap with its neighbors and thus, reducing its dynamic quenching. Phe16→Ala and Trp37→Leu mutations largely decreased the ability of NC to affect the local dynamics of 2Ap at positions 2 and 5, respectively, while a fingerless NC was totally ineffective. The restriction of 2Ap local mobility was thus associated with the NC hydrophobic platform at the top of the folded fingers. Since this platform supports the NC chaperone properties, the restriction of the local mobility of the bases is likely a mechanistic component of these properties.

Characterization of the Inhibition Mechanism of HIV-1 Nucleocapsid Protein Chaperone Activities by Methylated Oligoribonucleotides

“…The equilibrium binding constants of the GU-and GT-rich ONs at 100 mM NaCl (Table 2) were close to the reported affinities of NC to TG-or UG-containing hexanucleotides or dodecanucleotides (2,3,43), suggesting that the GU or GT motifs provide a major contribution to NC binding to such ONs. Interestingly, an increase of the ionic strength only moderately reduced the affinities of the GU-and GT-rich ONs but caused a dramatic drop for the random sequence (Table 2, Fig.…”

Flexible Nature and Specific Functions of the HIV-1 Nucleocapsid Protein

“…92 In addition to the hydrophobic interactions, the V13, F16, T24 (I24), and A25 side chains in the hydrophobic plateau on the top of the ZFs interact with 10-UG-11 in the 8-GGUG-11 loop of SL2 and 8-AG-9 in the 6-GGAG-9 loop of SL3 to stabilize the complex. NCp7 was also found to bind with high affinity to single-stranded TG, GNG, and TNG motifs [94][95][96][97][98][99][100] (Table 1). These motifs are specifically recognized by NCp7 in two viral (−) DNA SLs, namely, the (−)primer binding site (PBS) and the (14-39)complementary trans-activation response element (cTAR).…”

Biophysical studies of the nucleic acid chaperone properties of the HIV-1 nucleocapsid protein

“…Recent observations showed that NCp7 can "freeze" the DNA. 80,81 As a consequence, the binding of NCp7 results in a loss of entropy, due to the decrease in the number of available NA conformations. In this context, NCp7 can be referred to as a "guide" that funnels NAs toward their most stable conformations by multiple routes within the conformational hyperspace.…”