ABSTRACT The mechanism by which the ammonium transporter, AmtB, conducts NH1
4 =NH3 into the cytoplasm was investigated
using conventional molecular dynamics (MD) simulations. These simulations revealed that the neutral molecule, NH3,
passes automatically through the channel upon its arrival at the Am2 site and that the function of the channel as a one-way
valve for passage of NH3 is not determined by the cytoplasmic exit gate but, rather, by the periplasmic entrance gate of the
channel. The NH3, produced by deprotonation of NH1
4 at the entrance gate, is spontaneously conveyed to the central region of
the channel via a hydrogen-bond network comprising His-168, His-318, Tyr-32, and the NH3 molecule. Finally, the NH3 molecule
exits the channel through the exit gate formed by Phe-31, Ile-266, Val-314, and His-318. In addition, Ser-263 is shown to play a
critical role in the final stages, acting as a pivoting arm to shunt the NH3 molecule from the cytoplasmic exit gate of the channel
out into the cytoplasm. This finding is further supported by another simulation which shows that NH3 fails to be translocated
through the channel formed by the Ser-263CAla mutation. Thus, this study casts new insights on the mechanism of AmtBmediated
passage of NH3 across cellular membranes.