The Structure and Mechanism of the Na+/H+
Antiporter NhaA
Catherine A. Fair
Comprehensive Paper
The Catholic University of America
February 15, 2008
1
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
2
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+.
3
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
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
5
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
6
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
7
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
8
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
9
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
10
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
11
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
Site-directed mutagenesis was performed on G336L, F344A, A160L, A127L, G104L,
A100L, and F72A. These were accomplished using a QuickChange II XL Site-Directed
Mutagenesis Kit to create mutation primers. The activities of these mutated proteins were then
assayed to determine whether or not they were able to function well enough to keep the cells
alive in varying concentrations of NaCl. The bacteria were plated and grown. The light
scattering of each plate was proportional to and therefore indicative of the concentration of
12
Figure 7. The penetration of water into the binding site at Asp 164 in the deprotonation and
protonation states of Asp 163.2
bacteria present. Figure 8 shows the locations of the mutated sites in the upper diagram and the
arrows mark the substrate pathways from the cytoplasm and the periplasm. The lower six circles
show the concentration of bacteria grown at increasing levels of NaCl for the mutated and wildtype bacteria. 2
13
Figure 8. Results of mutagenesis experiments.2
The activities of the mutant proteins were also compared to the activities of wild-type
(wt) protein and negative control bacteria (pBR). The results showed that the A100L mutation
was the only one that could maintain life at 0.6 M of NaCl. The G336L mutation could not even
maintain function at the lowest of concentrations. The mutations of A127L and G104L sustained
life at low concentrations of NaCl but failed at higher concentrations. According to Arkin et al.,
this is “consistent with blockage of a water-mediated proton entry to Asp 163 from the
14
periplasm”2 indicating that these residues line a pathway to Asp 163 from the periplasm. The
mutation of A127L was studied particularly to provide images of water movement. The water
molecules (and therefore protons as well) had much greater access to Asp 163 and Asp 164 in
the wild type protein than in the A127L mutant. This indicates that Ala 127 lines the periplasmic
passageway to Asp 163. The mutation of A160L also inhibited growth, and Arkin et al. state that
this shows blockage of a pathway to Asp 163 from the cytoplasm. This shows that A160 lines
the cytoplasmic passageway to Asp 163. Therefore, there are two passageways to Asp 163, one
from the cytoplasm and one from the periplasm. 2
Mechanism of Na+/H+ Exchange
There are two slightly different explanations to be found for the mechanism of Na+/H+
exchange. Hunte et al. describe an “alternating access mechanism.” They first describe the
structure of the antiporter when it is in an inactive configuration at low pH. As shown in Figure
9a, the cation binding site Asp 164 is only partially open to the cation pathway. A barrier that
includes helix XIp blocks the periplasmic pathway. When the pH sensor recognizes alkaline pH,
it causes a conformational change in helix IX which in turn causes the movement of helics XIp
and IVc to allow the whole binding site area, both Asp 163 and Asp 164, to be completely
accessible to the cytoplasmic pathway as shown in Figure 9b. At this point, the protein is active,
and instead of an ion barrier guarding the periplasmic pathway, the polypeptide chains
connecting XIc to XIp and IVc to IVp serve as the barrier. 3 At the start of the exchange, both
Asp 163 and Asp 164 are in a state of deprotonation and open to the cytoplasm. The binding of
the cation causes a charge imbalance which moves helices XIp and IVc and their connecting
polypeptide chains. This opens both Asp 163 and Asp 164 to the periplasm. Once the cation is
15
released, Asp 163 and Asp 164 become protonated, which causes a conformational change so
that both are accessible once more to the cytoplasm. Then they are both deprotonated and the
cycle is ready to start again.3
Figure 9. Conformational changes showing the activity of the antiporter.3
Arkin et al. also describe the mechanism, but their mechanism is significantly different
from that of Hunte et al. In the beginning of Arkin et al.’s description, the accessibility-control
site Asp 163 is deprotonated. Asp 164 is protonated and open to the cytoplasm. The cation
binding site, Asp 164, then releases a proton into the cytoplasm and Na+ binds in its place. A
proton from the periplasm then protonates Asp 163 which causes a conformational change in the
protein, shown in Figure 10, so that the binding site Asp 164 is now accessible to the periplasm. 2
This is different from Hunte et al. who state that it is the binding of the cation that opens the
binding site to the periplasm.3
16
Figure 10. The conformational change that occurs with the protonation of Asp 163.
Next, the Na+ bound to Asp 164 is replaced with H+ from the periplasm. Arkin et al. hold
that this replacement “could be considered a ‘knock-on’-like mechanism acting through
electrostatic repulsion.”2 The term “knock-on” is used by Hodgkin and Keynes in a 1955 paper
in which they discussed the mechanism of ion transfer in a K+ channel. According to this
mechanism, K+ collisions on side 1 cause each K+ in the channel to move one place to the right
toward side 2. Eventually, the last K+ in the channel is released into side 2.5 Berneche and Roux
discussed the importance of ion-ion repulsion in such a mechanism. Figure 11 shows a diagram
of the channel at different stages of cation movement. The diagrams have different sites in the
channel labeled with S1, S2, etc. In the potassium channel, S1 was the outermost possible site of
an ion in the channel, and S4 was the innermost possible site of an ion in the channel.
Movement of ions was from the “inner cavity” on the left to “external” on the right. In this
model, the effect of the location of two colliding ions on the ion in site S1 was considered.
When one colliding ion was in the cavity and the other was at site S3, an energy barrier
prohibited the release of the outermost ion as illustrated in graph a of Figure 11. This was also
the case when one ion had moved into site S4 and the other was either in site S3 or between S3
17
and S2 (as seen in Figure 11 b and c). However, once the two colliding ions were in positions S4
and S2, the repulsion between the two cations was so strong, one ion was forced to depart, and
the ion in site S1 was released from the channel. This relieved the tension between the two
cations and lowered the energy. 6
Figure 11. Removal of energy barrier as attacking ions approach the ion to be removed.6
Therefore, it can be understood relative to the NhaA protein that the positive charge
repulsion between the proton and the sodium ion would force the sodium ion out of the binding
site so that the proton could take its place.
In the final step, Asp 163 is deprotonated which results in a conformational change that
opens the Asp 164 binding site to the cytoplasm again. Overall, one Na+ has moved from the
cytoplasm to the periplasm and two H+ have been moved from the periplasm to the cytoplasm. 2
18
An important point to note is that the protein can only change from one conformation to another
when it is bound to a substrate, either one cation or two protons.3 The overall mechanism as
described by Arkin et al. is illustrated in Figure 12.
Figure 12. The transportation mechanism of the NhaA antiporter according to Arkin et al.2
There are notable differences between these two mechanisms. First of all, Hunte et al.
provide a description of the inactive configuration which is absent from Arkin et al.’s
explanation. Next, while Hunte et al. said that Asp 163 and Asp 164 were always open to the
same side at the same time, Arkin et al. had found that the two residues were always open to
opposite sides, as was shown in Figure 7.2 Another significant difference is that in Hunte et al.’s
explanation, both Asp 163 and Asp 164 are always protonated or deprotonated at the same time.3
However, in Arkin et al.’s description, the protonation of the two residues takes place at different
times.2
19
Antiporter Substrates
The protein NhaA is known as the sodium-proton antiporter, but sodium is not the only
cation transported by the protein. Schuldiner and Fishkes examined five possible substrates for
the protein: sodium, potassium, guanidine (supposedly a replacement for sodium in nerves),
lithium, and choline. It had been observed that if a sodium gradient were set up across a
membrane, a pH gradient would be formed because the sodium ions were being transported.
Therefore, to test the efficiency of the five possible substrates, membrane vesicles were infused
with chloride salts of the test ions and the vesicles were examined for the presence of a pH
gradient which would indicate that transport of the test ion had taken place. The method of
measuring a pH gradient was based on the measurement of the fluorescence of 9-aminoacridine.
The fluorescence of 9-aminoacridine was observed to change when membranes containing
sodium ions were exposed to a substance lacking sodium ions, indicating the movement of
sodium ions. The fluorescence of 9-aminoacridine was observed to remain constant when such
membranes were exposed to a substance that contained sodium ions. Therefore, a large decrease
in fluorescence of 9-aminoacridine suggests that a large pH gradient has been formed across the
membrane of a vesicle due to the NhaA pump. 7
As shown in Figure 13, the presence of sodium was observed to cause a large change in
fluorescence of 9-aminoacridine. The presence of lithium also caused a large change in
fluorescence. Guanidine, choline, and potassium did not cause a change in fluorescence. This
indicates that NhaA is capable of transporting either sodium or lithium. 7
20
Figure 13. The changes in fluorescence due to the presence of various substrates to determine
the substrate of the antiporter.7
Padan and colleagues also investigated the substrate specificity of the sodium-proton
pump. They noted that E. coli cells that exhibited increased antiporter activity were able to
survive in much higher concentrations of Li+, indicating that they were resistant to Li+. Cells
that did not show increased antiporter activity remained sensitive to Li+ ions. The cells with high
antiporter activity were able to handle 100 mM of Li+ ions while wild type cells exhibiting
normal antiporter activity could only handle 10 mM of Li+ ions. The high Li+ resistance of these
cells has thus been attributed to the increased antiporter activity which was able to remove the
high number of toxic Li+ ions from the cell. Padan and his colleagues determined that the gene
ant coded for the Na+/H+ antiporter. They also determined that cells without a functional ant
gene exhibited decreased growth in the presence of increasing concentration of NaCl. This
sensitivity to high concentrations of Na+ without the presence of the ant gene supports the idea
21
that the same antiporter, coded by the ant gene can transport either Li+ or Na+ ions out of the
cell.8
Padan and his colleagues also found a separate K+/H+ antiporter. Unlike the Na+/H+
antiporter, this protein is capable of transporting Na+ or K+. They determined that these were
two separate antiporters because treatment with trypsin decreased the activity of the K+/H+
antiporter, but treatment with trypsin did not affect the activity of the Na+/H+ antiporter.8
Arkin et al examined the thermodynamics of substrate specificity of NhaA with free
energy perturbation calculations. They morphed Na+ to K+ in water and also in the binding site
of the protein. The difference in binding energies was calculated from the difference of the free
energies of morphing Na+ to K+ in water versus the difference of morphing the two in the
binding site. Figure 14 shows these energy differences. It was found that NhaA binds Li+ more
strongly than Na+ by 16 kJ/mol. However, K+ was found to bind to NhaA more weakly than Na+
by 14 kJ/mol. This gives a thermodynamic reason for the fact that NhaA is observed to have a
slightly greater affinity for Li+ than for Na+.2
Figure 14. The free energy of the binding of K+, Na+, and Li+ to the antiporter.2
22
The potentials of mean force were also calculated for Na+ and Li+. This is a method of
calculations that identifies a kinetic energy barrier. Figure 15 shows a graph of the potentials of
mean force versus the distance across the antiporter. The distance across the antiporter is
measured in terms of angstroms across the z-axis of the antiporter in three-dimensional space.
At the location of the binding site, -7.5 Ǻ on the z-axis, ∆G for both Li+ and Na+ is very close to
-1 kJ/mol. This indicates that both of these ions lack a significant kinetic barrier to reach the
binding site. However, K+ was not able to reach the binding site. Rather, K+ became stuck in
different local minima on the graph. Thus, the presence of a kinetic barrier in the approach of a
K+ ion to the binding site may also contribute to the selectivity of NhaA for Na+ and Li+ over
K+.2
Figure 15. The free energy of potentials of mean force for Li+ and Na+.2
23
Conclusion
The Na+/H+ antiporter NhaA helps maintain pH in the cell in addition to 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 whose activity decreases as pH becomes
increasingly acidic. Asp 133 is the key residue in the pH sensor. Asp 164 is the binding site.
When it is protonated, Na+ is released from the protein, and when Asp 164 is deprotonated, Na+
becomes bound to the protein. Asp 163 is the accessibility-control site. When it is protonated,
Na+ is released to the periplasm, and when Asp 163 is deprotonated, Na+ is released to the
cytoplasm. There are separate passageways to Asp 164 and Asp 163. Hunte et al. describe a
different mechanism of exchange than Arkin et al. do. In Hunte et al.’s explanation, there is a
description of the inactive configuration and both Asp 163 and Asp 164 are in the same state of
protonation and open to the same side at the same time. In Arkin et al.’s mechanism of
exchange, Asp 163 and Asp 164 are open to opposite sides and are protonated independently of
each other. They also explain that the replacement of Na+ with H+ is accomplished through a
“knock-on” mechanism which uses electrostatic repulsion. While Na+ is the primary substrate of
the antiporter, NhaA actually has a slightly greater affinity for Li+.
24
References
1
Berg, J. M., Tymoczo, J. L., Stryer, L. Biochemistry. Ed. 6th. W.H. Freeman and Company:
New York, 2007.
2
Arkin, I.; Xu, H.; Jensen, M.; Arbely, E.; Bennett, E.; Bowers, K.; Chow, E.; Dror, R.;
Eastwood, M.; Flitman-Tene, R.; Gregersen, B.; Klepeis, J.; Kolossváry, I.; Shan, Y.; Shaw, D.,
Science, 2007, 317, 799.
3
Hunte, C.; Screpanti, E.; Venturi, M.; Rimon, A.; Padan, E.; Michel, H., Nature, 2005, 435,
1197.
4
Rothman, A., Padan, E., Schuldiner, S., J. Biol. Chem, 1996, 271, 32288.
5
Hodgkin, A.L., Keynes, R.D., J. Physiol., 1955, 128, 61.
6
Berneche, S., Roux, B., Nature, 2001, 414, 73.
7
Schuldiner, S., Fishkes, H., Biochemistry, 1978, 17, 706.
8
Padan, E., Maisler, N., Taglicht, D., Karpel, R., Schuldiner, S., J. Biol. Chem., 1989, 264,
20297.
25