NhaA antiporter functions using 10 helices, and an additional 2 contribute to assembly/stability

Abstract

The Escherichia coli Na + /H + antiporter (Ec-NhaA) is the best-characterized of all pH-regulated Na + /H + exchangers that control cellular Na + and H + homeostasis. Ec-NhaA has 12 helices, 2 of which (VI and VII) are absent from other antiporters that share the EcNhaA structural fold. This α-hairpin is located in the dimer interface of the Ec-NhaA homodimer together with a β-sheet. Here we examine computationally and experimentally the role of the α-hairpin in the stability, dimerization, transport, and pH regulation of Ec-NhaA. Evolutionary analysis (ConSurf) indicates that the VI-VII helical hairpin is much less conserved than the remaining transmembrane region. Moreover, normal mode analysis also shows that intact NhaA and a variant, deleted of the α-hairpin, share similar dynamics, suggesting that the structure may be dispensable. Thus, two truncated Ec-NhaA mutants were constructed, one deleted of the α-hairpin and another also lacking the β-sheet. The mutants were studied at physiological pH in the membrane and in detergent micelles. The findings demonstrate that the truncated mutants retain significant activity and regulatory properties but are defective in the assembly/stability of the Ec-NhaA dimer.

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

“…8). Because of these structural differences163031 the dimerization interface in NhaA is less extensive than that seen in NapA. Consistently, our MS data showed that NhaA dimers were easily dissociated following desolvation, whereas NapA dimers were not.…”

Structure and function of yeast and fungal Na+/H+ antiporters

“…Helices 6 and 7 form an alpha hairpin at the dimer interface. Deletion of these TM segments causes defective dimerization, assembly, and stability, although the protein still retains significant transport activity . In Ec NhaA, a beta sheet on the periplasmic side of the membrane is also important in dimerization.…”

Molecular modeling and inhibitor docking analysis of the Na+/H+exchanger isoform one

“…There are dimer contacts with extracellular beta strands and TM 1, TM 9, TM 6 and TM 7. Helices 6 and 7 form an alpha hairpin at the dimer interface (Padan et al 2015). In EcNhaA, a beta sheet on the periplasmic side of the membrane is also important in dimerization (Rimon et al 2007).…”

“…Assignment of these residues as a helix has been contentious in the past with one model assign them as a helix (Wakabayashi et al 2000) and another suggesting that they form two separate TM segments (TM7 and TM8) (Landau et al 2007). This region is weakly conserved among Na + /H + antiporters (Dutta and Fliegel 2018) and the corresponding region in NhaA of E. coli is located at the dimer interface (Padan et al 2015). When mutated to Cys, amino acid 351 of this region is accessible to extracellular sulfhydryl labeling.…”

“…Na + /H + antiporters have been shown to consist of two distinct domains: a dimerization or scaffolding domain and a helix bundle or transportation domain (Hendus-Altenburger et al 2014;Padan et al 2015). The NHE1 model possesses an inverted topology of transmembrane helices as reported (Hendus-Altenburger et al 2014;Padan et al 2015) for other Na + /H + exchangers. It is arranged such that TM2-TM3-TM4 and TM8-TM-9 are intertwined to make the dimerization domain whereas TM5-TM6-TM-7 and TM10-TM11-TM12 form the metal transportation domain (Fig.…”