Background:  The tenofovir (TDF) -associated mutation K70E is increasing in prevalence and was observed in virologic non-responders of the abacavir/lamivudine/tenofovir arm of the ESS30009 study. K70E has not been observed in the same viral clone as the TDF-associated mutation K65R, and site-directed mutants with K65R+K70E have very poor replication capacity. We employed molecular dynamic simulations to uncover the structural basis of the antagonism between these mutations.

Methods:  Stochastic boundary molecular dynamic simulations used the starting RT-dTTP structure 1RTD and the CHARMM program to simulate the motions of atoms within a 20-Ǻ sphere from the ligand. A 10 ns simulation was performed for each variant (wild type, R65, E70, and R65+E70) and results for the last 5 ns trajectories were analyzed.

Results:  Root mean square deviations between the molecular dynamic models and the 1RTD structure were small (0.92 Ǻ to 1.4 Ǻ). The e-amino group of K65 is positioned 2.74±0.12 Ǻ from the γ-phosphate of the dTTP ligand and serves to stabilize the triphosphate moiety. In the R65 mutant, the distance between the guanidino side chain and the g-phosphate increases to 4.19±0.39 Ǻ and the interaction energy between residue 65 and the ligand is +55 kcal/mol less favorable. However, the K70 e-amino group is repositioned to partially compensate for this destabilization by moving 2.4 A closer to the γ-phosphate (3.51±0.57 Ǻ) and has a –67 kcal/mol more favorable interaction energy with the ligand. The negatively charged E70 in the double mutant cannot provide compensatory stabilization of the γ-phosphate, resulting in a more severe defect. The γ-phosphate is stabilized in by an intramolcular hydrogen bond with the 3’-OH of the ribose moiety of the ligand. The hydrogen bond (distance <3.5 Ǻ) is present in 56 to 69% of the wild type and single-mutant trajectories but only 14% of the trajectories for the double mutant.

Conclusions:  Atomic interactions that stabilize the terminal pyrophosphate leaving group of the dNTP ligand are lost in both R65 and R65+E70 mutants. A novel compensatory interaction between K70 and the γ-phosphate of the ligand appears in the single mutant but is lost in the double mutant, resulting in a more severe defect. The loss of an intramolecular hydrogen bond to the γ-phosphate may further destabilize the pyrophospate leaving group in the double mutant. Additional molecular dynamic simulations of dideoxy analogs may help elucidate this effect.