194 Energetics and dynamics of adenine protonation in HIV-1’s dimerization initiation site

Abstract

HIV-1, like most retroviruses, packages two homologous copies of its RNA genome. The two RNA strands are non-covalently linked near their 5’ends. The proposed dimerisation initiation site is a 35-nucleotide (nt) stem loop capable of forming loop–loop interactions (a kissing dimer) via its highly conserved 6-nt loop palindrome. In a structural transformation affected by temperature, salt concentration, and by the HIV-1 nucleocapsid protein, the initial, kinetically-stable kissing dimer (KD) converts to a thermodynamically-stable extended dimer. It has been suggested that this in vitro observed rearrangement is associated with the in vivo viral genome maturation. Mihailescu et al. demonstrated enhanced rearrangement dynamics triggered by the protonation of a specific adenine residue at the genome sequence location 272 (A272) (Mihailescu, 2004). They suggested that the local environment of A272 caused its N1 atom’s pKa to shift upwards (more basic) by approximately 2.5 pH units when compared with 5’-adenosine monophosphate (5’-AMP). In this work, we investigated the dynamics and energetics of protonating A272’s N1 atom with explicit solvent molecular dynamics (MD) simulations, Thermodynamic Integration (TI), and the Poisson–Boltzmann equation (PB). Two initial structures were used, an NMR solution structure (PDB ID: 2D19) and an X-ray crystal structure (PDB ID: 1XP7), where A272 was found inside (NMR) and outside (X-ray) the helical axis respectively. MD simulations showed when A272 started,it remained inside the KD’s axis, and when outside, it attempted to insert itself within the axis. Calculated pKa shifts obtained from solving the PB equation were approximately + 2.2 and + 1.0 pH units for the NMR and X-ray structures respectively; while TI calculations performed with the NMR structure yielded a shift of approximately + 2.5 pH units. Our simulations confirmed the strong influence of A272's local environment on its calculated pKa when inserted in the helical axis. A272's N1 atom was approximately 200 times more likely protonated when compared with 5'-AMP. Also, protonated A272s were more energetically favoured in the kissing dimer versus an isolated monomer due to a diminished positive electrostatic potential near A272, while in the dimer. Overall, our computational investigations affirm the experimental suggestion that A272 in the kissing dimer is more likely to be protonated at physiological conditions and this protonation may trigger the structural rearrangement of the initial kissing dimer to form the extended dimer.