Deprotonation by Dehydration: The Origin of Ammonium Sensing in the AmtB Channel

The AmtB channel passively allows the transport of NH(4) (+) across the membranes of bacteria via a “gas” NH(3) intermediate and is related by homology (sequentially, structurally, and functionally) to many forms of Rh protein (both erythroid and nonerythroid) found in animals and humans. New structural information on this channel has inspired computational studies aimed at clarifying various aspects of NH(4) (+) recruitment and binding in the periplasm, as well as its deprotonation. However, precise mechanisms for these events are still unknown, and, so far, explanations for subsequent NH(3) translocation and reprotonation at the cytoplasmic end of the channel have not been rigorously addressed. We employ molecular dynamics simulations and free energy methods on a full AmtB trimer system in membrane and bathed in electrolyte. Combining the potential of mean force for NH(4) (+)/NH(3) translocation with data from thermodynamic integration calculations allows us to find the apparent pK(a) of NH(4) (+) as a function of the transport axis. Our calculations reveal the specific sites at which its deprotonation (at the periplasmic end) and reprotonation (at the cytoplasmic end) occurs. Contrary to most hypotheses, which ascribe a proton-accepting role to various periplasmic or luminal residues of the channel, our results suggest that the most plausible proton donor/acceptor at either of these sites is water. Free-energetic analysis not only verifies crystallographically determined binding sites for NH(4) (+) and NH(3) along the transport axis, but also reveals a previously undetermined binding site for NH(4) (+) at the cytoplasmic end of the channel. Analysis of dynamics and the free energies of all possible loading states for NH(3) inside the channel also reveal that hydrophobic pressure and the free-energetic profile provided by the pore lumen drives this species toward the cytoplasm for protonation just before reaching the newly discovered site.