deprotonation by dehydration the origin of ammonium sensing in the amtb channel去质子化的脱水铵传感的起源amtb通道.pdfVIP

Deprotonation by Dehydration: The Origin of Ammonium Sensing in the AmtB Channel David L. Bostick1,2, Charles L. Brooks III1,2* 1 Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, United States of America, 2 Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, United States of America The AmtB channel passively allows the transport of NH4þ across the membranes of bacteria via a ‘‘gas’’ NH3 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 NH4þ 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 NH3 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 NH4þ/NH3 translocation with data from thermodynamic integration calculations allows us to find the apparent pKa of NH4þ 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 bindi