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Regulation of NhaA by Protons

Etana Padan

10.1002/cphy.c100078

Source: Volume 1, Issue 4, October 2011

Published online: October 2011

Full Article on Wiley Online Library



Abstract

H+, a most common ion, is involved in very many biological processes. However, most proteins have distinct ranges of pH for function; when the H+ concentration in the cells is too high or too low, protons turn into very potent stressors to all cells. Therefore, all living cells are strictly dependent on homeostasis mechanisms that regulate their intracellular pH. Na+/H+ antiporters play primary role in pH homeostatic mechanisms both in prokaryotes and eukaryotes. Regulation by pH is a property common to these antiporters. They are equipped with a pH sensor to perceive the pH signal and a pH transducer to transduce the signal into a change in activity. Determining the crystal structure of NhaA, the Na+/H+ antiporter of Escherichia coli have provided the basis for understanding in a realistic rational way the unique regulation of an antiporter by pH and the mechanism of the antiport activity. The physical separation between the pH sensor/transducer and the active site revealed by the structure entailed long‐range pH‐induced conformational changes for NhaA pH activation. As yet, it is not possible to decide whether the amino acid participating in the pH sensor and the pH transducer overlap or are separated. The pH sensor/transducer is not a single amino acid but rather a cluster of electrostatically interacting residues. Thus, integrating structural, computational, and experimental approaches are essential to reveal how the pH signal is perceived and transduced to activate the pH regulated protein. © 2011 American Physiological Society. Compr Physiol 1:1711‐1719, 2011.

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Figure 1. Figure 1.

Properties of NhaA. (A) ΔpH‐driven 22Na+/H+ antiporter activity at pH 8.5 and (B) passive efflux (ΔpH and ΔΨ = 0 by the presence of acetate and valinomycin/KCl) as a function of pH were measured in proteoliposomes containing purified NhaA. Experimental details in 57.
Figure 2. Figure 2.

General architecture of NhaA Na+/H+ antiporter 22. (A) Ribbon representation of the crystal structure viewed parallel to the membrane (broken line). The 12 transmembranes (TMs) are labeled with Roman numerals. The cytoplasmic and periplasmic funnels are marked (black line). Cytoplasmic or periplasmic‐oriented TMs are denoted as c and p, respectively. (B) Functional organization in NhaA. A stick‐and‐ball representation of functionally important residues in the putative active site (red or black) and the pH sensor (yellow or magenta). (C) The TMs IV‐XI assembly with the interrupted TMs and the putative active site (black circle).
Figure 3. Figure 3.

NhaA mutants with different response to pH. Everted membrane vesicles were prepared from the bacterial strain EP432 expressing the indicated mutations. The Na+/H+ antiporter activity was determined at the indicated pH values using Acridine orange fluorescence to monitor ΔpH in the presence of 10 mM (continuous line) or 100 mM (broken line) NaCl as described in 11,12. The results are expressed as end level of dequenching.
Figure 4. Figure 4.

Clusters of amino acid residues in NhaA with strongly electrostatic interaction as revealed in silico by multiconformation continuum electrostatics (MCCE) analysis. Cluster I (magenta) cluster II (white) cluster III (yellow), and cluster IV (green) are shown. Zones of negative and positive potential are colored in red and blue, respectively. This figure was prepared after 34.
Figure 5. Figure 5.

Conformational change in NhaA at K249 identified by trypsin digestion. Purified His‐taged NhaA was subjected to trypsin digestion at the indicated pH values and run on SDS‐PAGE. The pH profile of NhaA Na/H antiport and the trypsin digestion are shown in blue and green. For further details see 14.


Figure 1.

Properties of NhaA. (A) ΔpH‐driven 22Na+/H+ antiporter activity at pH 8.5 and (B) passive efflux (ΔpH and ΔΨ = 0 by the presence of acetate and valinomycin/KCl) as a function of pH were measured in proteoliposomes containing purified NhaA. Experimental details in 57.



Figure 2.

General architecture of NhaA Na+/H+ antiporter 22. (A) Ribbon representation of the crystal structure viewed parallel to the membrane (broken line). The 12 transmembranes (TMs) are labeled with Roman numerals. The cytoplasmic and periplasmic funnels are marked (black line). Cytoplasmic or periplasmic‐oriented TMs are denoted as c and p, respectively. (B) Functional organization in NhaA. A stick‐and‐ball representation of functionally important residues in the putative active site (red or black) and the pH sensor (yellow or magenta). (C) The TMs IV‐XI assembly with the interrupted TMs and the putative active site (black circle).



Figure 3.

NhaA mutants with different response to pH. Everted membrane vesicles were prepared from the bacterial strain EP432 expressing the indicated mutations. The Na+/H+ antiporter activity was determined at the indicated pH values using Acridine orange fluorescence to monitor ΔpH in the presence of 10 mM (continuous line) or 100 mM (broken line) NaCl as described in 11,12. The results are expressed as end level of dequenching.



Figure 4.

Clusters of amino acid residues in NhaA with strongly electrostatic interaction as revealed in silico by multiconformation continuum electrostatics (MCCE) analysis. Cluster I (magenta) cluster II (white) cluster III (yellow), and cluster IV (green) are shown. Zones of negative and positive potential are colored in red and blue, respectively. This figure was prepared after 34.



Figure 5.

Conformational change in NhaA at K249 identified by trypsin digestion. Purified His‐taged NhaA was subjected to trypsin digestion at the indicated pH values and run on SDS‐PAGE. The pH profile of NhaA Na/H antiport and the trypsin digestion are shown in blue and green. For further details see 14.

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Article Title: Regulation of NhaA by Protons
 

How to Cite

Etana Padan. Regulation of NhaA by Protons. Compr Physiol 2011, 1: 1711-1719. doi: 10.1002/cphy.c100078