One of the phosphate groups present in the large ribosomal subunit of Haloarcula marismortui (P2485) is highly buried in the active site. Its interactions with the protonated forms of A2486 and of the substrate N-term moiety-two positive species that could be involved in the enzymatic mechanism-are here investigated by molecular simulation methods. Molecular dynamics calculations are first used to construct fully hydrated structural models of the active site in the free state and in complex with a model substrate. Density functional and electrostatic calculations based on the Poisson-Boltzmann equation based on these models suggest that the phosphate moiety significantly stabilizes the protonated form of the substrate and, moreover, that of A2486 by long-range electrostatic interactions. In contrast, our calculations provide no evidence for the previously proposed stabilization of A2486 via a charge-relay mechanism.