Structure-based Functional Study Reveals Multiple Roles of Transmembrane Segment IX and Loop VIII–IX in NhaA Na+/H+ Antiporter of Escherichia coli at Physiological pH

Abstract

The three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na ؉ /H ؉ antiporter. However, because NhaA is activated at physiological pH (pH 6.5-8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII-IX. Here we studied this NhaA segment (Glu 241 -Phe 267 ) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H ؉ /Na ؉ stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na ؉ , suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu 255 and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10 -18 Å ) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-Å cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent K m for Na ؉ of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation.Regulation of intracellular pH, cellular Na ϩ content, and cell volume is essential for all living cells. Na ϩ /H ϩ antiporters play primary roles in these crucial processes. They are integral membrane proteins, ubiquitous throughout the biological kingdom. Many Na ϩ /H ϩ antiporters are tightly regulated by pH, a property that underpins their capacity to maintain pH homeostasis of the cytoplasm (1).NhaA, the main Na ϩ /H ϩ antiporter of Escherichia coli, has eukaryotic orthologs, including human (2, 3). It is an electrogenic antiporter with a stoichiometry of 2 H ϩ /1 Na ϩ (1, 4) and is strongly dependent on pH; its rate of activity changes over 3 orders of magnitude between pH 7.0 and 8.5 (1, 4, 5).NhaA is a dimer in the native membrane as revealed by genetic complementation data, biochemical pulldown experiments, intermolecular cross-linking (6), ESR studies (7,8), and cryoelectron microscopy of two-dimensional crystals (9, 10). The recently determined crystal structure of NhaA monomer at pH 4 (11) has provided the first structural insights into the mechanism of antiport and pH regulation of a Na ϩ /H ϩ antiporter. NhaA consists of 12 TMSs 2 with the N and C termini pointing into the cytoplasm. It represents a novel fold; TMSs IV and XI are each comprised ...

β-Sheet-dependent Dimerization Is Essential for the Stability of NhaA Na+/H+ Antiporter

“…In a recent study (27), we used scanning Cys replacements for intermolecular cross-linking and identified amino acid residues that participate in the NhaA dimer interface at the cytoplasmic side of the membrane: (i) Residues Ser 246 and Pro 247 at the junction between TMS IX and loop VIII-IX and (ii) L255 inside TMS IX. As predicted by the electron spin resonance based model of the NhaA dimer (14), the residues that cross-link efficiently cluster on one face of TMS IX, Table S1, Fig.…”

“…Mutants P247C, A248C,and L255C were described previously in Ref. 27. The double mutant ⌬(P45-N58)-L255C was obtained on a pCL-AXH3 background by ligating the BglII-MluI fragment (682 bp) with BglII-MluI fragment (4.08 kb) of plasmid pCL-AXH3.…”

“…As described previously, residues in the N terminus of TMS IX and in loop VIII-IX (Ser 246 , Pro 247 , Ala 248 , Val 254 , and Leu 255 ) form the interface of the NhaA dimer at the cytoplasmic side of the membrane (27). To test whether these residues are important in maintaining the NhaA dimer, we studied the mobility of Cys replacements P247C, A248C, and L255C on BN-PAGE (Fig.…”

Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT

“…The 10 single-cysteine variants of sodium:proton antiporter NhaA TM9 are known to form dimers without cross-linking reagents, and the positions forming dimers are located at the N-terminal half of NhaA TM9 (43). Pulsed electron paramagnetic resonance distance measurements of NhaA demonstrated that the TM9s of each protomer of NhaA are close to each other (22).…”

Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter

“…4b). Our previous MTSET accessibility test of Cys replacements in TMS IX also showed that the cytoplasmic funnel deepens into the membrane at physiological pH as compared with acidic pH (48).…”