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.

HIV-1 retroviral genomic RNA dimerization is initiated by loop-loop interactions between the SL1 stem-loops of two identical RNA molecules. The SL1-SL1 unstable resulting kissing complex (KC) then refolds irreversibly into a more stable complex called extended dimer (ED). Although the structures of both types of complex have been determined, very little is known about the conformational pathway corresponding to the transition, owing to the difficulty of observing experimentally intermediate conformations. In this study, we applied targeted molecular dynamics simulation techniques (TMD) to the phosphorus atoms for monitoring this pathway for the backbone, and a two-step strategy was adopted. In a first step, called TMD(-1), the dimer structure was constrained to progressively move away from KC without indicating the direction, until the RMSD from KC reaches 36A. A total of 20 TMD(-1) simulations were performed under different initial conditions and different simulation parameters. For RMSD ranging between 0 and 22A, the whole set of TMD(-1) simulations follows a similar pathway, then divergences are observed. None of the simulations leads to the ED structure. At RMSD=22A, the dimers look like two parallel Us, still linked by the initial loop-loop interaction, but the strands of the stems (the arms of the Us) are positioned in such a manner that they can form intramolecular as well as intermolecular Watson-Crick base-pairs. This family of structure is called UU. In a second step (TMD simulations), 18 structures were picked up along the pathways generated with TMD(-1) and were constrained to move toward ED by decreasing progressively their RMSD from ED. We found that only structures from the UU family are able to easily reach ED-like conformations of the backbones without exhibiting a large constraint energy.

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.

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.

Base pairing at the stem-loop junction in the SL1 kissing complex of HIV-1 RNA: a thermodynamic study probed by molecular dynamics simulation.

The thermodynamics of the opening/closure process of a GC base pair located at the stem-loop junction of the SL1 sequence from HIV-1(Lai) genomic RNA was investigated in the context of a loop-loop homodimer (or kissing complex) using molecular dynamics simulation. The free energy, enthalpy and entropy changes for the closing reaction are 0 kcal x mol(-1), -11 kcal x mol(-1) and -0.037 kcal x mol(-1) x K(-1) at 300 degrees K respectively. Furthermore it is found that the free energy change is the same for the formation of a 11 nucleotide loop closed with UG and for the formation of a 9 nucleotide loop closed with GC. Our study evidences the high flexibility of the nucleotides at the stem-loop junction explaining the weak stability of this structure.