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

Tzvi Tzubery, Abraham Rimon, Etana Padan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

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 (Glu241-Phe267) 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 Leu255 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 Km for Na+ of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation.

Original languageEnglish
Pages (from-to)15975-15987
Number of pages13
JournalJournal of Biological Chemistry
Volume283
Issue number23
DOIs
StatePublished - 6 Jun 2008

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