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 A) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-A 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.
Highlights
NhaA, the main Naϩ/Hϩ antiporter of Escherichia coli, has eukaryotic orthologs, including human [2, 3]
The results presented here fully support the structural model of the NhaA dimer and show that in addition to the -hairpin at the periplasmic side of NhaA a segment in TMS IX and loop VIII–IX contains amino acid residues that are located at or in proximity to the NhaA dimer interface
Our results show that chemical modification of L255C at the NhaA dimer interface dramatically affects the activity of NhaA and its pH regulation: the larger the chemical modifier (AMS), the greater the inhibitory effect on the Naϩ/Hϩ antiporter activity
Summary
Bacterial Strains and Culture Conditions—EP432 is an E. coli K-12 derivative, which is melBLid, ⌬nhaA1::kan, ⌬nhaB1::cat, ⌬lacZY, thr1 [13]. The beads were washed twice in binding buffer [26] at pH 7.4, resuspended in 100 l of binding buffer containing 0.2 mM fluorescein-5-maleimide (Molecular Probes), and further incubated for 30 min at 25 °C to determine the Cys left free following MTSET treatment. Treatment of Membranes Expressing Cys Replacement Mutations with SH Reagents for Activity Assay—Membranes (0.5 mg of membrane protein) were resuspended in a reaction mixture as described above for cross-linking but containing one of the following reagents: 1 mM NEM, 1 mM MTSES (Anatrace), 1 mM MTSET (Anatrace), 1 mM AMS (Molecular Probes), or a 4 mM concentration of the oxidizing reagent diamide. All experiments were repeated at least twice with practically identical results
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