Mechanism of Na+/H+ Antiporting

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Science  10 Aug 2007:
Vol. 317, Issue 5839, pp. 799-803
DOI: 10.1126/science.1142824


Na+/H+ antiporters are central to cellular salt and pH homeostasis. The structure of Escherichia coli NhaA was recently determined, but its mechanisms of transport and pH regulation remain elusive. We performed molecular dynamics simulations of NhaA that, with existing experimental data, enabled us to propose an atomically detailed model of antiporter function. Three conserved aspartates are key to our proposed mechanism: Asp164 (D164) is the Na+-binding site, D163 controls the alternating accessibility of this binding site to the cytoplasm or periplasm, and D133 is crucial for pH regulation. Consistent with experimental stoichiometry, two protons are required to transport a single Na+ ion: D163 protonates to reveal the Na+-binding site to the periplasm, and subsequent protonation of D164 releases Na+. Additional mutagenesis experiments further validated the model.

NhaA is the archetypal Na+/H+ antiporter and the only member of the family that is absolutely required by E. coli for survival in high-salt conditions, under alkaline stress, or in the presence of otherwise toxic Li+ concentrations (1, 2). It is a membrane protein consisting of 388 residues that traverse the inner membrane 12 times, with both termini ending in the cytoplasm (3). The structure of NhaA exhibits a distinctive fold of 10 contiguous transmembrane helices and 2 antiparallel, discontinuous helices (iv and xi) aligned end to end to span the membrane (4).

NhaA excretes Na+ or Li+ (but not K+) from the cytoplasm using the energy from the cotransport of protons down their electrochemical gradient into the cell, with a characteristic electrogenic stoichiometry of two protons to one Na+ or Li+ (5, 6). NhaA's activity decreases by three orders of magnitude when shifting from pH 8 to pH 6.5 (7), enabling it to regulate cellular acidity in addition to cellular salinity.

Using recently developed algorithms for the high-speed, parallel execution of molecular dynamics (MD) simulations (811), we performed simulations of membrane-embedded NhaA with an aggregate length approaching 3 μs, allowing us to examine ion transport, pH regulation, and cation selectivity and to thereby deduce a detailed mechanistic picture of the function of NhaA.

Crystallization of E. coli NhaA was performed at pH 4, a state in which the protein is inactive, and accordingly, no Na+ was seen in the structure (4). Hunte et al. (4) inferred, however, that the Na+-binding site is near D163 or D164 (12), because these have been shown to be the only carboxylic residues absolutely indispensable for transport activity (13). Because these residues are likely candidates to undergo protonation state changes that drive Na+ transport, we simulated NhaA under all four possible combinations of the protonation states of D163 and D164. After equilibration, we initiated MD simulations (12 to 100 ns each) from each of these four configurations, with Na+ placed adjacent to D163. We then repeated the process, placing Na+ adjacent to D164.

When Na+ was placed adjacent to D163, irrespective of the protonation state of D163 or D164, the ion became trapped by the protein, and water was unable to penetrate the protein to hydrate it; this finding suggests that D163 is not the Na+-binding site. The behavior of Na+ was entirely different when placed adjacent to D164. If D164 was deprotonated, Na+ remained bound regardless of the protonation state of D163, with water able to reach and hydrate the ion. Placing Na+ adjacent to protonated D164, however, provided the strongest clues as to how the transporter might function. In this case, the ion was expelled from the protein to the aqueous environment, with the direction of Na+ release determined by the protonation state of D163. If D163 was protonated, Na+ was expelled to the periplasm; in contrast, if D163 was deprotonated, Na+ was expelled to the cytoplasm. (See Fig. 1A.)

Fig. 1.

Na+ transport and alternating access. D163 and D164 are colored in blue and red, respectively. (A) Series of Na+ (purple) positions when D163 (not shown) is deprotonated or protonated, leading to Na+ expulsion to the cytoplasm or transport to the periplasm, respectively. The rightmost panel depicts Na+ position as a function of time in different D163 and D164 protonation states. (B) The four graphs show water penetration into the occupied Na+-binding site at D164 (top graphs) and the regulatory site at D163 (bottom graphs) as a function of position (z) across the membrane. The average density and SD were computed from two 60-ns trajectories, with D163 protonated (left) and deprotonated (right). Blue and red lines in graphs represent positions of D163 and D164, respectively. The top panel shows representative snapshots from the simulations, with water molecules shown in pink and white. (C) Mutagenesis studies of NhaA. Wild-type (WT) and mutated antiporters were assayed at different NaCl concentrations. Negative control bacteria (pBR) expressed no antiporter. The top panel shows locations of the different mutation sites (green) in the NhaA structure. Substrate pathways from the cytoplasm and periplasm are indicated by blue arrows. Molecular images in Figs. 1, 2, and 4 and Fig. S1 were produced by VMD (29), and Fig. 1 and Fig. S3 were generated by PyMOL (30).

We therefore propose that D163 and D164 play different roles in the Na+ transport mechanism. D164 is the binding site for Na+: Its protonation state determines whether Na+ binds to the protein or is released. When D164 is deprotonated and, consequently, negatively charged, Na+ remains tightly bound as a result of Coulombic attraction. When D164 is protonated, the electrostatic interaction is diminished and Na+ no longer binds. In contrast, D163 is the accessibility-control site of the protein: Its protonation state is the molecular switch that determines whether the Na+-binding site (i.e., D164) is accessible to the periplasm or to the cytoplasm. Bound Na+ can be expelled to the periplasm if D163 is protonated or to the cytoplasm if D163 is deprotonated; in each case, expulsion is accompanied by protonation of D164.

Our simulations provide a structural explanation for how D163 acts as a conformational switch. The carboxylate group of deprotonated D163 makes strong hydrogen bonds with the amide hydrogens of residues M105 and T132 (Fig. 2). Upon protonation, these interactions vanish, and D163 forms a new hydrogen bond between its carboxylic hydrogen and the carbonyl oxygen of M105. This movement causes helix v to tilt, which shifts the Na+-binding site (D164) in the direction of the periplasm.

Fig. 2.

Effects of D163 protonation state on antiporter conformation. D164 is protonated, such that bound Na+ would be expelled. The top panels depict the local interaction of D163, whereas the bottom panels illustrate the global conformation of the protein. The structures in which D163 is protonated (orange) and deprotonated (white) are superposed by Cα atoms (D164 is protonated in both structures). The Cα root mean square deviation (RMSD) between the structures is ∼2.3 Å when comparing the central helices ii to v, ix, and xi.

The local structural changes elicited by D163 protonation lead to global conformational changes (Fig. 2). When D163 is protonated, the cytoplasmic entrance is closed while the periplasmic exit is open. Deprotonation of D163 leads to the closure of the periplasmic Na+ exit and the opening of the cytoplasmic entrance. Taken together, our data provide a molecular realization of the alternate accessibility mechanism postulated by Jardetzky more than 40 years ago (14).

The hypothesis that the protein contains a Na+/H+-binding site at D164 and a H+-binding site at D163 implies that protons and Na+ must be able to reach D164 and that protons must be able to reach D163. A putative Na+ transport pathway, notably blocked in the inactivated x-ray structure, has been suggested (4), starting from a large crevice on the cytoplasmic face of the protein, leading to D164, and ending in the periplasm. However, the existence of a pathway to D163 has not been previously considered.

To identify possible ion transport pathways, we determined the water accessibility of the two sites for different protonation states of the protein. Water can reach both D163 and D164 from both sides of the membrane (Fig. 1B), but in no protonation state is there a continuous water density across the protein; such water connectivity might deplete the proton motive force. Instead, when D163 is accessible to the cytoplasm, D164 is accessible to the periplasm (Fig. 1B, left panels), and when D163 is accessible to the periplasm, D164 is accessible to the cytoplasm (Fig. 1B, right panels).

We undertook mutagenesis experiments to substantiate our computational finding of these water pathways to D163. We hypothesized that replacing smaller amino acids with larger ones along these pathways would block access of protons to D163 and thus inhibit the antiporter. Activities of wild-type and mutant antiporters were assayed for their ability to sustain growth of bacteria that do not harbor any of the three native antiporter genes (15). Unlike the wild-type antiporters, all mutant antiporters aside from Ala100→Leu100 (A100L) failed to support growth in elevated concentrations of NaCl (Fig. 1C). Because the G336L mutant protein does not support bacterial growth even in a low concentration of NaCl, it is possible that this protein may be unstable and nonfunctional. The functional comparisons between the mutant antiporters were conducted in vivo, and the concentrations of the different proteins may thus differ despite identical handling.

The inhibitory effects of mutations at A127 and G104 are consistent with blockage of a water-mediated proton pathway to D163 from the periplasm, whereas the inhibitory effect of a mutation of A160 is consistent with blockage of a water-mediated proton entry to D163 from the cytoplasm. Moreover, we explicitly simulated the effect of mutating A127 to leucine (fig. S3) and found that water and, therefore, proton penetration from the periplasm to D163 are indeed blocked. Additional mutagenesis results are reported in the supporting online material (SOM).

Based on these results, we propose a mechanism for the Na+/H+ antiporting cycle of NhaA that consists of the following four sequential steps (labeled as in Fig. 3): (A) The accessibility-control site (D163) is deprotonated, resulting in cytoplasmic accessibility of the Na+-binding site. The Na+-binding site (D164) therefore releases a H+ to, and takes up a Na+ from, the cytoplasm. (B) The accessibility-control site (D163) is protonated by a H+ that enters from the periplasm. This results in a conformational change (Fig. 2) that exposes the Na+-binding site to the periplasm. (C) The Na+-binding site (D164) exchanges the bound Na+ with a H+ from the periplasm. The replacement of bound Na+ by H+ could be considered a “knock-on”–like mechanism (16) acting through electrostatic repulsion (17). (D) D163 deprotonates, leading to a protein conformational change that again exposes the Na+-binding site (D164) to the cytoplasm. Overall, two protons are taken up from the periplasm in panels B and C and released to the cytoplasm in panels A and D (Fig. 3), while a single Na+ is taken up from the cytoplasm in panel A and then pumped to the periplasm in panel C, which is consistent with NhaA's electrogenic stoichiometry of one Na+ to two protons (5).

Fig. 3.

Schematic representation of the transport model of NhaA. The carboxylic group of D163 in the accessibility-control site is colored blue, and D164 in the Na+-binding site is red.

Very recent simulations of NhaA (18) have explored conformational changes associated with the simultaneous deprotonation of a group of residues (not including D133, discussed below), but a detailed understanding of the mechanism of its pH sensitivity and the identity of its pH sensor has remained elusive. Although the pH activation range of NhaA closely resembles the pKa of histidine (where Ka is the acid dissociation constant), no single histidine is required for Na+ transport or is part of the pH sensor (19, 20). We thus focused instead on carboxylic residues as possible pH sensors, because some carboxylic residues are known to have highly elevated pKa's (21, 22). Recently, pKa's of all ionizable residues were calculated for acid-inactivated NhaA (23). Six carboxylic residues (D78, E82, E124, D133, D163, and D164) were found to have abnormally high pKa's, with D133 having the highest value. Having already conducted simulations with protonated D163 and D164, we performed four additional simulations (6 ns each): In each of these additional simulations, either D78, E82, E124, or D133 was protonated (with D163 and D164 protonated in all cases). Protonating the “real” pH sensor should lead the protein to adopt a structure similar to the x-ray structure, which was determined at low pH (4), conditions under which the pH sensor is presumably protonated.

Our simulations suggest that D133 is a key component of the pH sensor. D133 resides between the N termini of helix iv and helix xi, thereby neutralizing their opposing helical dipoles (Fig. 4). Upon protonation of D133, the two opposing helices shift such that their dipoles no longer converge on a single point, because a protonated carboxylic group cannot effectively neutralize the opposing dipoles. This conformation is similar to the acid-inactivated x-ray structure (4). Moreover, among those carboxylic residues exhibiting elevated pKa's (23), D133 was the only one whose protonation resulted in such a conformational change.

Fig. 4.

Effect of D133 protonation on NhaA structure. The leftmost and middle panels display the structures resulting from simulations in which D133 was deprotonated or protonated. The rightmost panel displays the x-ray crystal structure obtained at pH 4 (4). Due to the resolution of the x-ray analysis (3.45 Å), no hydrogen is seen in the structure. Red arrows highlight the positions of the helical axes. The helix coloring is as described in fig. S1. The change from the x-ray structure in a simulation with D133 protonated is noticeably smaller than the corresponding change when D133 is deprotonated (Cα RMSD of 2.0 Å versus 2.5 Å when comparing helices ii, iv, v, and xi).

Mechanistically, the shift of the two helical dipoles observed in our simulations upon D133 protonation causes D164 in the Na+-binding site to face the protein lumen rather than the Na+ entry and exit pathways (Fig. 4). In this position, D164 is unable to bind Na+, resulting in an inactive protein. D164 also faces the protein lumen in the low-pH x-ray structure, further pointing to D133 as the pH sensor. Mutation of D133 to the neutral residue asparagine has been shown experimentally (13, 24) to result in nearly complete inhibition of transport, similar to the inactivation caused by lowering of pH. Moreover, mutation of G338, located at the juncture between the helical dipoles, markedly reduces the pH response of the transporter (25).

The antiporter must be, and is (6), extremely selective for Na+ over K+, and is slightly selective for Li+ over Na+ (26, 27). To investigate NhaA's selectivity among different cations, we conducted free-energy perturbation calculations (28) and found that NhaA (i) binds Li+ more strongly than Na+ (by 16 kJ/mol) and (ii) binds K+ more weakly than Na+ (by 14 kJ/mol) (fig. S4A). Potential of mean force analyses (28) show that neither Na+ nor Li+ encounters a substantial kinetic barrier to binding (fig. S4B). Our results indicate that NhaA's ion selectivity can be explained thermodynamically.

Our combinatorial approach, based on multiple long–time-scale MD simulations, led to the formulation of a model of NhaA's transport mechanism, pH regulation, and cation selectivity that is consistent with both our own and previously reported experimental data. Future studies will be needed to (i) identify the exact source of coupling between Na+ and proton transport, which is necessary to avoid proton leakage by NhaA, and (ii) determine whether mammalian Na+/H+ exchangers, which differ in their Na+:H+ stoichiometry and pH response, use a transport mechanism similar to that proposed for NhaA.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S4

Tables S1 to S4


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