The crossing of two unwound transmembrane regions that is the hallmark of the NhaA structural fold is critical for antiporter activity.

Cell pH and Na homeostasis requires Na/H antiporters. The crystal structure of NhaA, the main Escherichia coli Na/H antiporter, revealed a unique NhaA structural fold shared by prokaryotic and eukaryotic membrane proteins. Out of the 12 NhaA transmembrane segments (TMs), TMs III-V and X-XII are topologically inverted repeats with unwound TMs IV and XI forming the X shape characterizing the NhaA fold.

Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters.

Na/H antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine.

Insight into the direct interaction of Na with NhaA and mechanistic implications.

Na/H antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life that are essential in cellular ion homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystals provided insight in the structure of this molecular machine. However, structural data revealing the composition of the binding site for Na (or its surrogate Li) is missing, representing a bottleneck in our understanding of the correlation between the structure and function of NhaA.

An angular motion of a conserved four-helix bundle facilitates alternating access transport in the TtNapA and EcNhaA transporters.

There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, respectively.

Cardiolipin is an Optimal Phospholipid for the Assembly, Stability, and Proper Functionality of the Dimeric Form of NhaA Na/H Antiporter.

Cardiolipin (CL) was shown to bound to the dimer interface of NhaA Na/H antiporter. Here, we explore the cardiolipin-NhaA interaction both in vitro and in vivo. Using a novel and straightforward in-vitro assay in which n-dodecyl β-D maltoside (DDM) detergent is used to delipidate the dimer interface and to split the dimers into monomers; the monomers are subsequently exposed to cardiolipin or the other E.

Replacement of Lys-300 with a glutamine in the NhaA Na/H antiporter of yields a functional electrogenic transporter.

Much of the research on Na/H exchange has been done in prokaryotic models, mainly on the NhaA Na/H-exchanger from (EcNhaA). Two conserved aspartate residues, Asp-163 and Asp-164, are essential for transport and are candidates for possible binding sites for the two H that are exchanged for one Na to make the overall transport process electrogenic. More recently, a proposed mechanism of transport for EcNhaA has suggested direct binding of one of the transported H to the conserved Lys-300 residue, a salt bridge partner of Asp-163.

Broad phylogenetic analysis of cation/proton antiporters reveals transport determinants.

Cation/proton antiporters (CPAs) play a major role in maintaining living cells' homeostasis. CPAs are commonly divided into two main groups, CPA1 and CPA2, and are further characterized by two main phenotypes: ion selectivity and electrogenicity. However, tracing the evolutionary relationships of these transporters is challenging because of the high diversity within CPAs.

Asp133 Residue in NhaA Na/H Antiporter Is Required for Stability Cation Binding and Transport.

Na/H antiporters have a crucial role in pH and Na homeostasis in cells. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and revealed a previously unknown structural fold, which has since been identified in several secondary active transporters. This unique structural fold is very delicately electrostatically balanced.

Ligand-induced conformational dynamics of the Na/H antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry.

Na/H antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive.

Lysine 300 is essential for stability but not for electrogenic transport of the NhaA Na/H antiporter.

Na/H antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na, and volume. The NhaA antiporter of is the best studied member of the Na/H exchanger family and a model system for all related Na/H exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H ions that NhaA exchanges for one Na ion during one transport cycle.

The Ec-NhaA antiporter switches from antagonistic to synergistic antiport upon a single point mutation.

The Na(+), Li(+)/H(+) antiporter of Escherichia coli (Ec-NhaA) maintains pH, Na(+) homeostasis in enterobacteria. We used isothermal titration calorimetry to perform a detailed thermodynamic analysis of Li(+) binding to Ec-NhaA and several of its mutants. We found that, in line with the canonical alternative access mechanistic model of secondary transporters, Li(+)/H(+) binding to the antiporter is antagonistically coupled.

Sodium-Proton (Na(+)/H(+)) Antiporters: Properties and Roles in Health and Disease.

The transmembranal Na(+)/H(+) antiporters transport sodium (or several other monovalent cations) in exchange for H(+) across lipid bilayers in all kingdoms of life. They are critical in pH homeostasis of the cytoplasm and/or organelles. A particularly notable example is the SLC9 gene family, which encodes Na(+)/H(+) exchangers (NHEs) in many species from prokaryotes to eukaryotes.

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

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 Ec-NhaA structural fold. This α-hairpin is located in the dimer interface of the Ec-NhaA homodimer together with a β-sheet.

Overexpression, Isolation, Purification, and Crystallization of NhaA.

Living cells are critically dependent on processes that regulate intracellular pH, Na(+) content, and volume. Na(+)/H(+) antiporters play a primary role in these homeostatic mechanisms. They are found in the cytoplasmic and intracellular membranes of most organisms from bacteria to humans and have long been human drug targets.

NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential.

pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA.

Functional and structural dynamics of NhaA, a prototype for Na(+) and H(+) antiporters, which are responsible for Na(+) and H(+) homeostasis in cells.

The crystal structure of down-regulated NhaA crystallized at acidic pH4 [21] has provided the first structural insights into the antiport mechanism and pH regulation of a Na(+)/H(+) antiporter [22]. On the basis of the NhaA crystal structure [21] and experimental data (reviewed in [2,22,38] we have suggested that NhaA is organized into two functional regions: (i) a cluster of amino acids responsible for pH regulation (ii) a catalytic region at the middle of the TM IV/XI assembly, with its unique antiparallel unfolded regions that cross each other forming a delicate electrostatic balance in the middle of the membrane. This unique structure contributes to the cation binding site and allows the rapid conformational changes expected for NhaA.

Differential effects of mutations on the transport properties of the Na+/H+ antiporter NhaA from Escherichia coli.

Na(+)/H(+) antiporters show a marked pH dependence, which is important for their physiological function in eukaryotic and prokaryotic cells. In NhaA, the Escherichia coli Na(+)/H(+) antiporter, specific single site mutations modulating the pH profile of the transporter have been described in the past. To clarify the mechanism by which these mutations influence the pH dependence of NhaA, the substrate dependence of the kinetics of selected NhaA variants was electrophysiologically investigated and analyzed with a kinetic model.

The unwound portion dividing helix IV of NhaA undergoes a conformational change at physiological pH and lines the cation passage.

pH and Na(+) homeostasis in all cells requires Na(+)/H(+) antiporters. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and their pH regulation. Functional studies of NhaA in the membrane have yielded valuable information regarding its functionality in situ at physiological pH.

Conformational changes in NhaA Na+/H+ antiporter.

Na(+)/H(+) antiporters play a primary role in Na(+)/H(+) homeostasis in cells and many organelles and have long been drug targets. The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs (Trans membrane segments) are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site. Remarkably, inverted repeats containing interrupted helices with similar functional implications have since been observed in structures of other bacterial secondary transporters with almost no sequence homology.

Regulation of NhaA by protons.

H(+), a most common ion, is involved in very many biological processes. However, most proteins have distinct ranges of pH for function; when the H(+) concentration in the cells is too high or too low, protons turn into very potent stressors to all cells. Therefore, all living cells are strictly dependent on homeostasis mechanisms that regulate their intracellular pH.

Helix VIII of NhaA Na(+)/H(+) antiporter participates in the periplasmic cation passage and pH regulation of the antiporter.

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na(+)/H(+) antiporter. However, because NhaA is active at physiological pH (pH 6.5-8.

Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na+/H+ antiporter NhaA.

NhaA, a Na(+)/H(+) antiporter critical for pH and Na(+) homeostasis in Escherichia coli, as well as other enterobacteria and possibly Homo sapiens, was modified for fluorescence spectroscopy by constructing a functional Trp-less NhaA mutant. Purified Trp-less NhaA lacks the Trp fluorescence emission characteristic of the wild type, thereby providing a background for studying structure-function relationships in NhaA by site-directed Trp fluorescence. Two single-Trp variants in the Trp-less background (F136W and F339W) were constructed.