Periplasmic vestibule plays an important role for solute recruitment, selectivity, and gating in the Rh/Amt/MEP superfamily

Abstract

AmtB, a member of the Rh/Amt/MEP superfamily, is responsible for ammonia transport in Escherichia coli. The ammonia pathway in AmtB consists of a narrow hydrophobic lumen in between hydrophilic periplasmic and cytoplasmic vestibules. A series of molecular dynamics simulations (greater than 0.4 μs in total) were performed to determine the mechanism of solute recruitments and selectivity by the periplasmic vestibule. The results show that the periplasmic vestibule plays a crucial role in solute selectivity, and its solute preferences follow the order of NH4(+) > NH3 > CO2. Based on our results, NH4(+) recruitment is initiated by its interaction with either E70 or E225, highly conserved residues located at the entrance of the vestibule. Subsequently, the backbone carbonyl groups at the periplasmic vestibule direct NH4(+) to the conserved aromatic cage at the bottom of the vestibule (known as the Am1 site). The umbrella sampling simulations suggest that the conserved residue D160 is not directly involved in the ammonia conduction; rather its main function is to keep the structure of periplasmic vestibule intact. The MD simulations also revealed that two partially stacked phenyl rings of F107 and F215, separating the periplasmic vestibule from the hydrophobic lumen, flip open and closed simultaneously with a frequency of approximately 10(8) flipping events per second. These results show how the periplasmic vestibule selectively recruits NH4(+) to the Am1 site, and also that the synchronized flipping of two phenyl rings potentially facilitates the solute transition from the periplasmic vestibule to the hydrophobic lumen in the Rh/Amt/MEP superfamily.

Fig. 1.

(A) The dashed arrows show two observed paths for the recruitment of by periplasmic vestibule of AmtB (E225-A220-S219-F161 and E70-F68-G149-F161). (B) Probability distributions of substrates along the channel and at the vicinity of trimeric AmtB. The Y axis is the probability distribution (%) and the X axis is the channel coordinate. The curves in black, red, green, and blue correspond to , NH₃, CO₂, and H₂O distributions, respectively. Each point on the water distribution curve is the result of averaging over 10 sets of 100 water molecules. The blue error bars show the standard deviations at each point. The vestibule limits are marked with dashed lines. The arrow indicates the location of Am1 site. (C) ΔΔG along the Am transport axis that is calculated based the difference in SMD free energy of conduction relative to that of NH₃. The red dashed line shows where in the pathway the ΔΔG increases beyond 3.76 kcal/mol (the required energy for lowering the pK_a of ammonia to 6.5), which is the region between phenyl rings of F107 and F215. The energy profiles for and NH₃ conductions are shown in Fig. S7.

Fig. 2.

(A) The free energy profile of CO₂ conduction through AmtB channel calculated using umbrella sampling methods. The energy profile is the average value for three monomers. The regions between the two phenyl rings of F107/F215 are marked by a red rectangle. (B) Free energy profiles of conduction through native AmtB (red), “neutral D160” with constraints on the atomic distances (Blue), and “neutral D160” with no constraints (Green). (Inset) The four hydrogen bonds between D160 and the residues G163, G164, and T165. (C) The partially stacked phenyl rings of F107 and F215 flip open (Right) and close (Left) simultaneously. The red arrows show the directions of rotations. (D) The distribution of the torsion angles C_α-C_β-C_γ-C_δ1 for F107 (Left) and F215 (Right). For each residue the torsion angles corresponding with the closed and open states are indicated by red and blue arrows, respectively.