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First published online May 15, 2009
Journal of Experimental Biology 212, 1593-1603 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026708
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NhaA crystal structure: functional–structural insights

Etana Padan*, Lena Kozachkov, Katia Herz and Abraham Rimon

Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel


Figure 1
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  		Fig. 1. General architecture of NhaA Na+/H+ antiporter. (A) Ribbon representation of the crystal structure of NhaA, viewed parallel to the membrane (broken lines). The 12 transmembrane segments (TMSs) are labeled with roman numerals. N and C indicate the N- and C-termini, respectively. Cytoplasmic and periplasmic funnels are marked by continuous black lines; c or p denotes helices in the cytoplasmic or periplasmic sides, respectively. (B) Cross-section through the antiporter, normal to the membrane, with the front part removed. The cytoplasmic and periplasmic funnels are marked by white lines. Down to the white arrow, hydrated cations are potentially accessible into the cytoplasmic funnel. Below the red arrow, only non-hydrated Na+ or Li+ can potentially enter. The location of D164 is marked by a green star. For further details, see Hunte et al. (Hunte et al., 2005Go).

 

Figure 2
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  		Fig. 2. Distances in the crystal structure are compared with those obtained by intra-molecular cross-linking. The overall architecture of NhaA is shown as in Fig. 1A. Transmembrane segments (TMSs) IVp and TMSs XI of the TMSs IV–XI assembly are marked green. Segments of the TMSs lining the cytoplasmic funnel (II, IVc, V and IX) are marked yellow. Residues in the NhaA dimer interface (in the β-sheet and the N-terminus of TMS IX) are marked red. Arrows and numbers denote distances between indicated amino acids residues. The membrane is depicted by the broken lines. The representation was generated using PyMOL software (http://pymol.sourceforge.net/).

 

Figure 3
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  		Fig. 3. Model structure of NhaA dimer based on ESR study. Overview of the NhaA dimer seen from the periplasmic side and shown in ribbon representation was reproduced, with permission, from Hilger and colleagues (Hilger et al., 2007Go).

 

Figure 4
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  		Fig. 4. Discontinuous membrane helices in transport proteins. Crystal structures of transport proteins with discontinuous helices. (A) Ca2+-ATPase (Toyoshima et al., 2000Go). (B) NhaA (Hunte et al., 2005Go). (C) LeuT (Yamashita et al., 2005Go). (D) ClC (Dutzler et al., 2002Go). (E) GltT (Yernool et al., 2004Go). (F) SGlT (Faham et al., 2008Go). The left side of each panel shows the whole molecule (green) with the two discontinuous membrane helices colored in red and yellow according to their respective N- and C-termini. The right side of each panel shows an enlarged view of the discontinuous helices in cylindrical presentation. The partial charges at the termini are indicated. The estimated membrane position is shown (broken lines).

 

Figure 5
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  		Fig. 5. Functional organization of NhaA Na+/H+ antiporter. (A) Cylindrical representation of functionally important NhaA transmembrane segments (TMSs) viewed parallel to the membrane (broken lines). A stick and ball representation of functionally important residues in the putative active site (red) and the `pH sensor' (magenta) is shown. Residues in which cys-replacements affect only the pH-dependence of NhaA activity or both the pH-dependence and the apparent KM for Na+ are marked in yellow or magenta, respectively. The figure was prepared using PyMOL (http://pymol.sourceforge.net/). (B) Clusters of residues with strongly electrostatic interaction in NhaA as revealed in silico by multiconformation continuum electrostatics (MCCE) analysis (Olkhova et al., 2006Go). Four clusters of residues, which interact in a strong electrostatic manner cross NhaA along its cross-membrane axis: I, magenta; II, gray; III, yellow; IV, green. These are important for ion translocation and the `pH sensor' of NhaA (see A). Zones of negative and positive potential are colored red and blue, respectively. For orientation, other residues are indicated in black. This figure was prepared after Olkhova et al. (Olkhova et al., 2006Go) using Grasp (Honig and Nicholls, 1995Go).

 

Figure 6
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  		Fig. 6. Proposed mechanism of NhaA, a pH-regulated Na+/H+ antiporter. A schematic presentation of the pH-induced conformational changes (A,B) and the conformational changes accompanying Na+ antiport (B,C) (A) Acidic pH-locked conformation. Ion translocation is prevented by the periplasmic ion barrier (transparent, orange colored zone and red bar) and by the only residue (D164) within the Na+-Li+-binding site (residues D164, D163, T132, D133) that is exposed to the cytoplasmic funnel (black dotted line and black circle). (B) Activation by alkaline pH induces conformational changes in the `pH sensor' at the N-terminal of helix IX (red circle), resulting in reorientation of helices IVp, XIp and X. The putative Na+-Li+-binding site is now exposed to the cytoplasmic funnel but is sealed off towards the periplasm (red bar). (C) Na+-Li+ binding results in opening of the periplasmic funnel, which is sealed off from the cytoplasm (red bar) and exposure of the active site to the periplasm. The cation is released. Protonation of D163 and D164 brings the antiporter back to the active conformation, open to the cytoplasm. For orientation, the NhaA dimer interface is marked red at the N-terminus of helix IX and the β-sheet.

 

Figure 7
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  		Fig. 7. Mutagenesis studies in eukaryotic Na+/H+ exchangers (NHE) and NhaA Na+/H+ antiporter. The 3-D model structure of NHE1 is displayed with a gray ribbon and the residues that were mutated are presented using space-filled atoms using colors to represent the experimental outcome: residues that were implicated in ion-translocation (P167, P168, S235, D238, P239, A244, L255, I257, V259, F260, G261, E262, N266, D267, T270, S351, E391, C421 and Y454) are colored red, residues that are involved in pH regulation (R180, R327, E330, R440, G455 and G456) in magenta, residues comprising the NHE-inhibitors binding-site (F161, F162, L163, E346 and G352) in green and unessential residues (C133, Q157, P178, E184, C212, E248, H250, L254, H256, S263, V269, V271, F322, H325, S359, N370, S387, S388, S390, T392, S401, T402, S406, N410, K438, K443, C477, Q495 and R500) are in yellow. (A) A top view from the cytoplasmic side of the membrane. (B) A side view parallel to the membrane whereas the intracellular side is facing upward; the transmembrane segments (TMSs) are numbered in roman numerals. (C) A view from the extracellular side. (D) The model structure of NHE1 guided mutagenesis of NhaA and identification of the binding site of 2-aminoperimidine (2-AP), an amiloride derivative that inhibits NhaA. Amino acid residues of which cys-replacements affect the sensitivity of NhaA to 2-AP are marked green. Naïve residues are marked yellow.

 

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