On the stability of different experimental dimeric structures of the SL1 sequence from the genomic RNA of HIV‐1 in solution: A molecular dynamics simulation and electrophoresis study

Abstract

SL1 is a stem-loop RNA sequence from the genome of HIV-1 thought to be the initiation site for the dimerization of the retroviral genomic RNA. The aim of this study is to check the stability in solution of different experimental dimeric structures available in the literature. Two kinds of dimer have been evidenced: an extended duplex looking like a double helix with two internal bulges and a kissing complex in which the monomers with a stem/loop conformation are linked by intermolecular loop-loop interactions. Two divergent experimental structures of the kissing complex from the Lai isolate are reported in the literature, one obtained from NMR (Mujeeb et al., Nature Structural Biology, 1998, Vol. 5, pp. 432-436) and the other one from x-ray crystallography (Ennifar et al., Nature Structural Biology, 2001, Vol. 8, pp. 1064-1068). A crystallographic structure of the Mal isolate was also reported (Ennifar et al., Nature Structure Biology, 2001, Vol. 8, pp. 1064-1068). Concerning the extended duplex, a NMR structure is available for Lai (Girard et al., Journal of Biomolecular Structure and Dynamics, 1999, Vol. 16, pp. 1145-1157) and a crystallographic structure for Mal (Ennifar et al., Structure, 1999, Vol. 7, pp. 1439-1449). Using a molecular dynamics technique, all these experimental structures have been simulated in solution with explicit water and counterions. We show that both extended duplex structures are stable. On the contrary, the crystallographic structures of the Lai and Mal kissing complexes are rapidly destabilized in aqueous environment. Finally, the NMR structure of the Lai loop-loop kissing complex remains globally stable over a 20 ns MD simulation, although large rearrangements occur at the level of the stem/loop junctions that are flexible, as shown from free energy calculations. These results are compared to electrophoresis experiments on dimer formation.

Conformational transitions of flanking purines in HIV‐1 RNA dimerization initiation site kissing complexes studied by CHARMM explicit solvent molecular dynamics

“…40 We have not seen any visible correlation between the dynamics of flanking bases and stem melting. Interestingly, such stem instability has been reported in AMBER simulation of kissing complexes performed by Aci et al 49 but not in our extensive AMBER simulations. The reason of this difference is presently not clear to us.…”

“…Bulged-in/out conformations, some flanking bases are bulged-in whereas others remain bulged-out, bases do not form inter-strand stacking; (left), A272, A273, A272* are bulged-in, whereas A273* is bulged-out (simulation F2); (right), A273 is bulged-in, A272* and A273* are bulged-out, A272 makes hydrogen bond across the strands to the phosphate oxygen of A273* (shown in green) (simulation B3). 49 showed destabilization of the X-ray kissing complexes (melting of the stems), not observed by Reblova et al 46 Concerning MD simulations of the Mujeeb's NMR structure for DIS-B, the Reblova's simulations revealed large scale rearrangements in very early stages of the run, indicating that the initial structure is unrelaxed. In contrast, Aci et al 49 reported a globally stable trajectory with rearrangements at the stem-loop junction.…”

“…49 showed destabilization of the X-ray kissing complexes (melting of the stems), not observed by Reblova et al 46 Concerning MD simulations of the Mujeeb's NMR structure for DIS-B, the Reblova's simulations revealed large scale rearrangements in very early stages of the run, indicating that the initial structure is unrelaxed. In contrast, Aci et al 49 reported a globally stable trajectory with rearrangements at the stem-loop junction. Some simulations were also carried out with the CHARMM force field, 50 utilizing Xray and NMR structures for kissing complex of Subtype B.…”

“…They also proposed the mechanisms of the conversion from the kissingloop complex into the extended duplex. 49,51,[54][55][56] Other RNA kissing motifs were also studied by means of MD, including H3 stem-loop of Moloney murine leukemia virus, 46 TAR elements of HIV 31,57 and kissing complex from 23 rRNA. 58 Due to inconsistency among the previously published results for simulations of DIS kissing complexes and availability of new experimental structures we were motivated to substantially extend theoretical studies of kissing complexes.…”

Conformational Pathway for the Kissing Complex→Extended Dimer Transition of the SL1 Stem-Loop from Genomic HIV-1 RNA as Monitored by Targeted Molecular Dynamics Techniques

“…In vivo experiments have also suggested that dimerization is a two-step process as a conformational rearrangement of the dimeric RNA incorporated in the virion is observed during maturation. 31 Although many structural studies have been devoted to SL1, [26][27][28][29][30][32][33][34] very little is known about the conformational pathway of the KC/ED transition. On the basis of model building from the X-ray structure of KC, it was suggested that the transition could occur via cleavage and cross-religation process at the level of purines located at the stem-loop junction without disruption of any existing WatsonCrick base-pairs in KC.…”