The Structure and Mechanism of the Na+/H+
Antiporter NhaA
Catherine A. Fair
Comprehensive Paper
The Catholic University of America
February 15, 2008
Abstract:
The Na+/H+ antiporter NhaA plays the important role of helping maintain pH in the cell as well as regulating the salinity of the cell. NhaA transports two protons into the cell down the pH gradient as it moves one Na+ ion out of the cell. NhA is a transmembrane protein consisting of 388 amino acids in twelve transmembrane segments. Antiporter activity decreases as pH becomes increasingly acidic, and at pH 4 the protein is inactive. The cytoplasmic passageway is negatively charged at its opening to attract the positively-charged sodium ion. The funnel becomes narrower as it goes deeper into the protein so that fully-hydrated cations cannot pass through. The periplasmic funnel is shallow, ending at Asp 65. Asp 163 is the pH sensor and Asp 164 is the sodium ion binding site. According to Arkin et al., only one of the residues Asp 163 and Asp 164 is protonated at a time. When Asp 163 is in a state of deprotonation and Asp 164 is protonated, the passageway to the binding site is open to the cytoplasm. Asp 164 is then deprotonated and a Na+ ion from the cytoplasm binds to the Asp 164 binding site. The replacement of H+ with Na+ is accomplished through a “knock-on” mechanism which uses electrostatic repulsion. When Asp 163 is protonated, the protein undergoes a conformational change which opens Asp 164 to the periplasm. The Na+ ion is then released into the periplasm and replaced with H+. The deprotonation of Asp 163 causes a conformational change which opens the binding site Asp 164 to the cytoplasm again. According to Hunte et al., both Asp 163 and Asp 164 are in the same state of deprotonation or protonation during the mechanism, and while the protonation of these two residues causes the binding site to open to the cytoplasm, it is the charge imbalance due to the newly-bound cation that causes the binding site to open to the periplasm. They also give an explanation for the structure when the antiporter is inactive, stating that the binding site’s passageway to the cytoplasm is only partially exposed. While Na+ is the primary substrate of the antiporter, NhaA actually has a slightly greater affinity for Li+.
Introduction
Physiological pH is usually close to 7.4.1 The Na+/H+ antiporter has the important role of helping to maintain pH in the E. coli cell as well as regulate the salinity of the cell. 2 An antiporter is a type of secondary transporter. These are proteins that facilitate active transport of ions in and out of the cell. The thermodynamically favorable flow of one ion down its concentration gradient is used to power the simultaneous thermodynamically unfavorable movement of another ion up its concentration gradient. Antiporters are secondary transporters that move two species in opposite directions, one into the cell and one out of the cell.1 The Na+/H+ antiporter, also known as NhaA, transports two protons into the cell down the pH gradient as it moves one Na+ ion out of the cell. In this way, the salinity and pH homeostasis of the cell is maintained.
Structure of NhaA
NhaA is a transmembrane protein in Escherichia coli that consists of a chain of 388 amino acids. 3 Originally, NhaA was thought to be composed of eleven transmembrane segments. However, as shown in Figure 1, work by Rothman, Padan, and Schuldiner revealed the presence of twelve transmembrane segments with both the amino and carboxy termini on the cytoplasmic side of the membrane. 4
Figure 1. The primary structure of NhaA.4
The size of the molecule is about 40 Ǻ x 45 Ǻ x 50 Ǻ. The secondary and tertiary structure of the protein can be seen in Figure 2. The periplasmic side of the protein is flat because it is comprised of loops that lie close to the membrane. Loop I-II contains short helix Ia, which includes residues 35-41. The next structure is an anti-parallel β-sheet which contains residues 45-48 in β1 and residues 55-58 in β2. This structure lies parallel to the lipid bilayer and contributes to the flat, rigid character of the periplasmic side of the protein. The cytoplasmic side of the protein is not nearly as flat. Instead, it contains flexible loops, and helices II, V, IX and XII extend into the cytoplasm. This creates an uneven edge on the cytoplasmic side of NhaA. In general, the cytoplasmic surface of the protein contains postively-charged residues while the periplasmic face contains negatively-charged residues. The exception to this is a funnel on the cytoplasmic side that has a few negatively-charged residues at its opening on the surface. The funnel ends in the middle of the protein when its path is blocked by chains from helices IV and XI. A periplasmic funnel is oriented toward the cytoplasmic funnel, but the two are not connected. 3
Figure 2. Two stereoviews of the secondary and tertiary structure of NhaA.3
There are several significant structural elements of the protein. There are 10 contiguous transmembrane helices.2 Of these, helices III and X are S-shaped, helix IX is bent, and helices VII and VIII are very short. However, the last two transmembrane segments, IV and XI, contain a structural formation different from the other ten helices. As shown in Figure 3, both contain two short helices connected by a polypeptide chain. The helix on the cytoplasmic side is denoted with “c” and the helix on the periplasmic side is denoted with a “p.” Transmembrane structure IVp contains residues 121-131, and IV c contains residues 134-143. Transmembrane structure XIc includes residues 327-336 while XIp includes residues 340-350. Thus, it is evident that the two structures have opposite orientations in the membrane. However, this conformation causes two thermodynamically unfavorable arrangements of charges. Figure 3 shows that the inside edges of the helices XIp and IVc are polar with a slightly positive charge while the inside edges of helices XIc and IVp are polar with a slightly negative charge. The first cause of thermodynamic instability is due to the presence of these polar tips in the hydrophobic core of a membrane because the mix of polar and non-polar hydrophobic molecules is thermodynamically unstable. The second cause of instability is the fact that the positively-charged amino termini of helices IVc and XIp face each other and the negatively-charged carboxy termini of helices XIc and IVp face each other. This causes a thermodynamically unfavorable conformation because molecules of the same charge repel each other. To stabilize the structure, Asp 133 is located between helices IVc and XIp to neutralize the two positive charges of the amino termini. The negative charges of the carboxy termini of helices IVp and XIc are stabilized by the presence of nearby Lys 300 on helix X. 3
Figure 3. The location of Asp 133 stabilizes helices XI and IV.3
The pH Sensor
NhaA’s activity is pH dependent. The activity of the antiporter decreases by three orders of magnitude when pH is changed from 8 to 6.5, and at pH 4, the protein is inactive. Through a series of investigations, it was determined that the key component of the pH sensor is residue Asp 133. NhaA has a pH activation range which resembles that of histidine, but it has been previously determined that no particular histidine is integral to the pH sensor. Some carboxylic acid residues have highly elevated pKa’s, so Arkin et al. next searched for the pH sensor among six carboxylic residues found to have unusually elevated pKa’s: Asp 78, Glu 82, Glu 124, Asp 133, Asp 163, and Asp 164. The residue with the highest pKa was Asp 133. Molecular dynamic simulations were performed on the structure for 6 ns each. In each simulation, a different residue was protonated, although Asp 163 and Asp 164 were protonated in every simulation. The conformations adopted by the protein were then compared to the x-ray structure of the protein determined at pH 4. At this pH, it was assumed that the pH sensor was protonated, so matching conformations would be an indication that the residue in question was indeed the pH sensor. 2 Because NhaA is down regulated with an increase of protons, at a very low pH, crystals are formed that are very ordered, so the structure of the protein can be determined. 3
Figure 4. The structure on the left shows the conformation of NhaA when Asp 133 is deprotonated. The structure in the middle shows the conformation when Asp 133 is protonated, and the structure on the right shows the x-ray structure of NhaA at pH 4.2
Residue Asp 133 is located between the amino termini of helix IV and helix XI where its negative charge serves to neutralize the opposing positive charges of each helix. However, when Asp 133 is protonated, it can no longer neutralize the positive charges of the amino termini, so helices IV and XI shift. This new conformation closely resembles that of the x-ray structure at pH 4, indicating that Asp 133 is the pH sensor. As further evidence that Asp 133 must be the pH sensor, the protonation of the other five selected residues caused no conformational changes. 2
The Accessibility-Control Site and the Binding Site
Arkin et al. also found that Asp 163 is the “accessibility-control site” 2 and Asp 164 is the binding site of NhaA. When Na+ was positioned close to Asp 163, the protein trapped the ion and water was not able to enter the protein and hydrate it. However, when Na+ was positioned close to Asp 164, water was able to enter the protein and access the ion. The protonation state of Asp 164 then determined what happened to the ion. When Asp 164 was deprotonated, Na+ became bound to the protein, and when Asp 164 was protonated, Na+ was released from the protein. The protonation state of Asp 163 determined whether Na+ was released to the cytoplasm or the periplasm. When Asp 163 was protonated, the ion was released into the periplasm, and when Asp 163 was deprotonated, the ion was released into the cytoplasm. This information shows Asp 163 to be the accessibility-control site and Asp 164 to be the binding site.2
Internal Passageways for the Substrates
Thus, Na+ and H+ must be accessible to Asp 164 while H+ must be accessible to Asp 163. It has been determined that there are separate passageways to Asp 163 and Asp 164. 2
The Passageway to Asp 164
There is a specific passageway for Na+ to reach the binding site at Asp 164. This funnel is negatively charged at its cytoplasmic opening so as to attract the positively-charged sodium. As shown in Figures 5 and 6, on one side of the cytoplasmic opening is Asp 11 and on the other side are Glu 78, Glu 82, and Glu 252. However, once an ion gets past these negative residues at the opening of the channel, the funnel no longer discriminates based on charge. Therefore, as shown in Figure 6, the funnel becomes narrower as it goes deeper into the protein where its lining consists of non-polar residues. Cations that are fully hydrated thus cannot travel to the end of the funnel which ends with the Asp 164 of helix V in the middle of the membrane. This significant location of Asp 164 emphasizes its importance in the binding of the cation. There are several other residues that are located nearby, such as Asp 163, but they are not accessible to this passageway.3
Figure 5. The cytoplasmic pathway and the periplasmic pathway. They are separated by the cream-colored portions of the helices.3
As shown in Figure 6, the periplasmic funnel is shallow and ends at Asp 65 on helix II.
Asp 65 is separated from Asp 164 by 16Ǻ of non-polar residues.
Figure 6. Cross-sectional view of the cytoplasmic (upper) passageway and the periplasmic (lower) passageway.3
The Passageway to Asp 163
Arkin and his colleagues examined the possible pathway for the transfer of protons to Asp 163 by measuring the water accessibility for both Asp 163 and 164 when Asp 163 was protonated or deprotonated. Figure 7 shows that water is accessible to both residues from both the cytoplasm and the periplasm, but never from both at the same time. That would create a continuous water density across NhaA which might cause the loss of the proton motive force.2 The proton motive force is the inherent energy found in the proton gradient. It is partly due to the charge gradient of the protons across the cells and partly due to the concentration gradient of protons across the cell.1 If there were a continuous water passageway through the cell, the proton gradient might be destroyed thus destroying the energy of the proton motive force due to this gradient.
Several mutational experiments were performed to further investigate this passageway to Asp 163. It was hypothesized that if small residues along the actual pathways were replaced with larger residues, then the protons would not be able to pass through and NhaA would be inhibited.2