Figure 1.
The AmtB Trimer/Membrane/Electrolyte System Setup
(Left) A top-down view of the simulated system alongside several periodic images. The simulation cell boundaries are drawn in black around the central image. Lipid headgroup phosphorus and nitrogen atoms are shown as orange spheres, and lipid chains are light blue lines. The AmtB trimer is colored by monomer.
(Right) A side view of the central simulation cell. Water is shown as red points, Cl− ions are shown as green spheres, and NH4+ ions are shown in a space-filling representation (nitrogen is blue and hydrogens are white).
Figure 2.
Partial Densities across the Simulation Cell
Shows the system structure as a function of the transport axis, z (z = 0 coincides with the center of mass of the trimer backbone).
(A) Major system components (lipid, water, and protein). The partial density of the phosphate group is shown as a reference for roughly identifying the boundaries of the membrane. A black, vertical dotted line is drawn at the position of the peak in each leaflet's phosphate density.
(B) NH4+ and Cl− densities. A black vertical dotted line is drawn at leaflet phosphate positions. The average concentration of NH4+ and Cl− in the bulk is “measured” to be 158 ± 5 mM, based on this plot. Cl− binding is enhanced on the cytoplasmic (z < 0) surface and inhibited on the periplasmic (z > 0) surface due to the “external” field created by the channel (dipole moment symbol) as seen from the Cl− density maxima at ∼±2.7 nm. NH4+ binding (see density maxima at ∼±1.7 nm) is not as seriously affected, although its local concentration at ∼±2.7 nm (around the Cl− binding positions) is enhanced near the periplasmic (compared with the cytoplasmic) membrane surface.
(C) NH4+ binding to the membrane surface involves penetration, dehydration, and hydrogen bonding with carbonyl and ester oxygens near the glycerol backbone of lipid molecules as seen in previous work [29].
Figure 3.
PMF for NH4+ Along the Transport Axis
In units of RT = 2.48 kJ/mol at 298 K. The profile was shifted such that it takes on a value of zero in the bulk. The origin is the same as in Figure 2, with periplasm (z > 0) and cytoplasm (z < 0) separated by the membrane (membrane phosphate positions are marked with vertical dotted lines). Am (de)protonation regions are marked with green vertical bars, whose widths give the uncertainty in the regions. NH3 binding sites are marked with red vertical bars. NH4+ binding sites are marked Am1 (z = 1.07 nm) and Am5 (z = −1.37 nm), along with an additional binding feature, circled in blue (z = 1.32 nm). The bound state of NH4+ at each of the three sites is shown in molecular detail (labeled panels). All specific interactions within each site are described in the text.
Figure 4.
PMF for NH3 Along the Transport Axis
In units of RT = 2.48 kJ/mol at 298 K (solid black line). The origin is the same as in Figure 2. Am (de)protonation regions are marked with green vertical bars, whose widths give the uncertainty in the regions, and NH4+ binding sites are marked with gray vertical bars. The PMF profile for NH3 (solid line) has been shifted such that its value coincides with that of NH4+ (dashed black line) at the equivalence ((de)protonation) regions (at the center of each green bar). Thus, the free-energy barrier for bringing NH4+ from site Am2 to the periplasmic equivalence point is ∼15 RT units. NH3 binding sites are marked Am2 (z = 0.17 nm), Am3 (z = −0.27 nm), and Am4 (z = −0.68 nm). The free-energy barrier for bringing NH3 (assuming single occupancy) from site Am4 to the cytoplasmic equivalence point is ∼10 RT units. The bound state of NH3 at each of the three sites is shown in molecular detail (bottom panel). All specific interactions within each site are described in the text.
Figure 5.
AmtB Is Structurally Invariant Regardless of Its Occupied State
Structural alignment of channels occupied solely at site Am2 (silver), Am3 (red), and Am4 (blue). The window shows a closeup of the aligned binding sites. Slight differences are seen between each structure, but are within positional fluctuations.
Figure 6.
Apparent pKa and Hydration of Am Along the Transport Axis
(A) Free-energy cycle used to determine pKa of NH4+ as a function of the transport axis. is the free energy for the alchemical reaction NH4+ → NH3 in the bulk, far from the membrane, and ΔG zdp is the free energy for the same reaction at a particular position, z, along the transport axis.
is the free energy to bring NH4+ from the bulk to a particular position, z, and
is the analogous quantity for NH3.
(B) Apparent pKa (pKaapp) for NH4+ as a function of z for the relevant region of the channel. The top panel shows a closeup of the portion of the bottom panel enclosed in the rectangular box drawn with dashed lines. A large dashed black line marks neutral pH in both panels. Vertical dotted lines (bottom panel) mark the average positions of lipid phosphate groups. As in Figures 3 and 4, gray bars represent NH4+ binding sites, red bars represent NH3 binding sites, and green bars represent (de)protonation regions. The periplasmic (de)protonation region is at z = 0.77 ± 0.08 nm, and the cytoplasmic (de)protonation region is at z = −1.13 ± 0.07 nm. See the text for an explanation of the uncertainties (width of the green bars) in these regions.
(C) Synopsis of binding sites and deprotonation regions generated by multiple structural alignment of AmtB channel structures singly bound at each binding site (a total of five structures taken from umbrella sampling simulations) and the X-ray structure [17]. Some transmembrane helices have been removed from the protein (orange) to allow visibility of the binding sites. The positions of the X-ray sites are shown as red spheres, and Am binding sites derived from our analysis are shown as either blue spheres (in the case of NH3) or blue spheres with white hydrogen atoms (in the case of NH4+). Any observed differences in the positions of the sites are within fluctuations. (De)protonation regions are marked in green. The periplasmic and cytoplasmic (de)protonation regions coincide with the phenyl groups of F107 and F31, respectively.
(D) Hydration of NH3 (diamonds with dashed line) and NH4+ (circles with solid line) as a function of the transport axis derived by combining statistics from free MD production runs and umbrella sampling runs. The hydration number is defined as the average number of water molecules falling within the first coordination shell of Am. The first coordination shell of Am was taken to be the position of the first minimum in the NAm − Owater pair correlation function (Rcoord = 0.362 nm, see Figure S4).
Table 1.
Gibbs Free Energy for Various Am Reactions Determined by Thermodynamic Integration
Figure 7.
NH4+ at the Site of Deprotonation
The configuration was taken from umbrella sampling simulations. NH4+ has full access to vestibular water, continuously connected to the bulk solution, to allow the escape of a proton to the bulk in the form of a hydronium ion.
Figure 8.
Am Deprotonation and Reprotonation
Events reconstructed from snapshots taken from umbrella sampling simulations. Important Phe residues (F107, F215, and F31) are shown in light green.
(A) Translocation of Am from site Am1 to site Am2.
(A1) NH4+ in a fully hydrated state with six coordinating water molecules.
(A2) NH4+ bound to site Am1.
(A3) NH4+ overcomes an ∼15 RT free-energy barrier to reach the deprotonation region (also shown in Figure 7). F107 rotates to allow passage, and NH4+ hydration shell is stripped of all but one water molecule.
(A4) After losing a proton, NH3 moves further toward site Am2, and all hydration is lost. F215 begins to rotate to allow passage. Here we notice a transient “flip” of the H168. This is only one of many conformational possibilities for the luminal His pair (H168 and H318). Different tautomeric states and conformations are also possible, but not represented here.
(A5) F107 and F215 are both rotated and parallel, as NH3 accepts a hydrogen bond from H168, which has “flipped” back to assume its original hydrogen-bonded conformation with H318. (6) F107 and F215 are once again horizontal and parallel, as NH3 occupies site Am2.
(B) Translocation of Am from site Am4 to site Am5.
(B1) NH3 occupies site Am4, donating a hydrogen bond to H318. F31 and S263 interact with water, protecting the hydrophobic lumen from hydration.
(B2) NH3 climbs an ∼10 RT barrier to approach the reprotonation region near F31, losing its hydrogen bond to H318, displacing F31, and moving toward water.
(B3) After gaining a proton from water, Am proceeds as NH4+ and gains hydrogen bonds with water and S263 (hydroxyl oxygen).
(B4) NH4+ becomes more hydrated as it moves toward site Am5.
(B5) NH4+ binds to site Am5, and hydrogen bonds are formed with (three to four) water molecules, S263 hydroxyl oxygen, and D313 carboxylate oxygen.
Figure 9.
Dynamics and Favorability of Multiply NH3–Occupied Luminal States
(A) State diagram of all possibly occupied states of the luminal NH3 sites. Each state is shown diagrammatically (from top to bottom, for a single state icon). (1) Site Am1 represented by a concave-up cusp with a green star, representing a reservoir of NH3. (2) sites Am2, Am3, and Am4 are represented by circles. (3) Am5 is represented by a concave-down cusp. Occupied (by NH3) luminal sites are represented by black dots (within the circles representing sites Am2–4). The free energy required for each reaction (arrows) is given in kJ/mol. Black values were determined from the PMF of Figure 4, red values were determined algebraically by completing the embedded thermodynamic cycles, and blue values were determined using thermodynamic integration techniques (see Figure S5 and surrounding discussion).
(B) A demonstration of NH3 dynamics (1 ns) in the lumen. Horizontal red bars represent sites (listed in decreasing value of z) Am2, Am3, and Am4, respectively. For the first 300 ps of the simulation, the NH3 molecules are restrained (in the z-dimension only) to their binding positions. When the restraint is released, NH3 is seen to behave dynamically in the lumen, and there is a strong tendency for NH3 to move toward the site closest to the cytoplasmic end of the channel, site Am4.
(C) A snapshot illustrating the tendency for NH3 to migrate toward site Am4. Here three NH3 molecules have migrated from a state where Am2, Am3, and Am4 are occupied, to a state where one molecule occupies Am3 and two molecules “fight” to occupy site Am4.
Figure 10.
AmtB Trimer System Equilibration
(A) Centers of mass of ionic species on each leaflet of the biomembrane as a function of time during equilibration. The center of mass of lipid phosphate is shown for reference. The origin in this plot is taken to be the corner of the simulation cell (as opposed to the center of mass of the AmtB trimer backbone as seen in other plots).
(B) Root mean square deviation (RMSD) after alignment of AmtB protein structures from the equilibration trajectory with the initial structure of the simulation.