Determination of the Mechanism of Selectivity and Ammonia Conduction By AmtB Using MD Simulations

Abstract

The transport of ammonia, fundamental to the nitrogen metabolism in all domains of life, is carried out by the Rh/Amt/MEP membrane protein superfamily. The first structure of this family (AmtB from E. coli) shows a pathway for ammonia that includes two vestibules connected by a long and narrow hydrophobic lumen. The accepted mechanism for AmtB is to recruit NH4+ and conduct neutral NH3 by deprotonation of NH4+ at the end of the periplasmic vestibule. We conducted several MD simulations (total of more than 0.3μs) using a model of trimeric AmtB embedded into POPE lipid bilayer to determine the mechanism of ligands conduction in the ammonia channels.To determine the AmtB's selectivity, we added 100 ligand molecules to our solvated protein-lipid system and conducted unconstrained MD simulations for each ligand. The probability distribution for each ligand along the normal of the lipid plane shows that the periplasmic vestibule prefers NH4+ over NH3 and CO2. Our long MD simulations reveal that two stacking phenyl rings of F107 and F215 (located at the bottom of the periplasmic vestibule) simultaneously flip open and close with a frequency of ∼108 flip-open events per second. The frequency of flip open/close events is independent of the presence of NH4+ at the vestibule. This indicates that the rate of this channel is controlled not only by the concentration gradient of ammonia but also by the frequency of phenyl rings open/close events.Our simulations show that D160 along with the aromatic rings are essential for recruitment of NH4+ at the phenyl rings gate. In each ammonia conduction cycle, NH4+ stays behind the gate long enough for the gate to flip open and let ammonium enters the lumen.