Olkhova, E., Kozachkov, L., Padan, E. & Michel, H. Combined computational and biochemical study reveals the importance of electrostatic interactions between the “pH sensor” and the cation binding site of the sodium/proton antiporter NhaA of Escherichia coli. Proteins 76, 548-559
Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. The crystal structure of NhaA has provided a basis to explore the mechanism of ion exchange and its unique regulation by pH. Here, the mechanism of the pH activation of the antiporter is investigated through functional and computational studies of several variants with mutations in the ion-binding site (D163, D164). The most significant difference found computationally between the wild type antiporter and the active site variants, D163E and D164N, are low pK(a) values of Glu78 making them insensitive to pH. Although in the variant D163N the pK(a) of Glu78 is comparable to the physiological one, this variant cannot demonstrate the long-range electrostatic effect of Glu78 on the pH-dependent structural reorganization of trans-membrane helix X and, hence, is proposed to be inactive. In marked contrast, variant D164E remains sensitive to pH and can be activated by alkaline pH shift. Remarkably, as expected computationally and discovered here biochemically, D164E is viable and active in Na(+)/H(+) exchange albeit with increased apparent K(M). Our results unravel the unique electrostatic network of NhaA that connect the coupled clusters of the "pH sensor" with the binding site, which is crucial for pH activation of NhaA.
[Show abstract] [Hide abstract] ABSTRACT: Sodium-proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium-proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium-proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium-proton antiport in NhaA. © 2014 Lee et al.0Comments 7Citations
- "...a water-mediated interaction between Lys300 and Asp163 was predicted for NhaA at high pH (Olkhova et al., 2009), a direct interactionFigure 8. Effect of breaking the Asp163–Lys300 salt bridge on the pK a of con..."9). Although a water-mediated interaction between Lys300 and Asp163 was predicted for NhaA at high pH (Olkhova et al., 2009), a direct interactionFigure 8. Effect of breaking the Asp163–Lys300 salt bridge on the pK a of conserved residues. (A) Distributions of pK a values estimated with PROPKA 3.1 (Søndergaard et al., 2011) from all MD simulations shown as violin plots.
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[Show abstract] [Hide abstract] ABSTRACT: Yersinia pestis, the bacterium that historically accounts for the Black Death epidemics, has nowadays gained new attention as a possible biological warfare agent. In this study, its Na⁺/H⁺ antiporter is investigated for the first time, by a combination of experimental and computational methodologies. We determined the protein's substrate specificity and pH dependence by fluorescence measurements in everted membrane vesicles. Subsequently, we constructed a model of the protein's structure and validated the model using molecular dynamics simulations. Taken together, better understanding of the Yersinia pestis Na⁺/H⁺ antiporter's structure-function relationship may assist in studies on ion transport, mechanism of action and designing specific blockers of Na⁺/H⁺ antiporter to help in fighting Yersinia pestis -associated infections. We hope that our model will prove useful both from mechanistic and pharmaceutical perspectives.0Comments 5Citations
- "... a ''pH sensor'', which was found to potentially include residues such as E78, E252, H253 and H256 . These residues are located at the N-terminal side of the cytoplasmic funnel not only at the Escher..."The Yersinia pestis NhaA comprises of twelve TMSs; TMSs IV (orange) and XI (purple) form the unique assembly exactly as at the Escherichia coli protein, the N-and C-termini are exposed to the cytoplasm (see below), a funnel opens into the cytoplasm and continues to the middle of the membrane, a shallower funnel opens to the periplasm and is separated from the cytoplasmic funnel by non-polar residues that act as a barrier, the periplasmic face of the protein is flat owing to structured loops and the cytoplasmic face is rough with flexible loops and a few helices that protrude into the cytoplasm. Regulation of the antiporting activity of Escherichia coli NhaA requires a ''pH sensor'', which was found to potentially include residues such as E78, E252, H253 and H256 . These residues are located at the N-terminal side of the cytoplasmic funnel not only at the Escherichia coli template but also at the suggested model for the Yersinia pestis NhaA.
[Show abstract] [Hide abstract] ABSTRACT: Na(+)/H(+) antiporters are integral membrane proteins that exchange Na(+) for H(+) across the cytoplasmic membrane and many intracellular membranes. They are essential for Na(+), pH and volume homeostasis, which are crucial processes for cell viability. Accordingly, antiporters are important drug targets in humans and underlie salt-resistance in plants. Many Na(+)/H(+) antiporters are tightly regulated by pH. Escherichia coli NhaA Na(+)/H(+) antiporter, a prototype pH-regulated antiporter, exchanges 2 H(+) for 1 Na(+) (or Li(+)). The NhaA crystal structure has provided insights into the pH-regulated mechanism of antiporter action and opened up new in silico and in situ avenues of research. The monomer is the functional unit of NhaA yet the dimer is essential for the stability of the antiporter under extreme stress conditions. Ionizable residues of NhaA that strongly interact electrostatically are organized in a transmembrane fashion in accordance with the functional organization of the cation-binding site, ;pH sensor', the pH transduction pathway and the pH-induced conformational changes. Remarkably, NhaA contains an inverted topology motive of transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found to vary in other ion-transport proteins. This novel structural fold creates a delicately balanced electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation. Based on the crystal structure of NhaA, a model structure of the human Na(+)/H(+) exchanger (NHE1) was constructed, paving the way to a rational drug design.0Comments 61Citations
- "...lusters are essential for pH signal transduction across the membrane and NhaA activation (Olkhova et al., 2009). Combining the MCCE studies with molecular dynamics (MD) simulations has revealed that a structura..."H256 provides an electrostatic connection between Clusters I, II and III, and D133 connects Clusters I and III. We suggest that these unique electrostatic interactions between the clusters are essential for pH signal transduction across the membrane and NhaA activation (Olkhova et al., 2009). Combining the MCCE studies with molecular dynamics (MD) simulations has revealed that a structural change must occur in few parts of NhaA whereas most helices do not change conformation (Fig. 2;Fig.
- [Show abstract] [Hide abstract] ABSTRACT: Protons dictate the charge and structure of macromolecules and are used as energy currency by eukaryotic cells. The unique function of individual organelles therefore depends on the establishment and stringent maintenance of a distinct pH. This, in turn, requires a means to sense the prevailing pH and to respond to deviations from the norm with effective mechanisms to transport, produce or consume proton equivalents. A dynamic, finely tuned balance between proton-extruding and proton-importing processes underlies pH homeostasis not only in the cytosol, but in other cellular compartments as well.0Comments 432Citations
- [Show abstract] [Hide abstract] ABSTRACT: Human NHA2 is a poorly characterized Na(+)/H(+) antiporter recently implicated in essential hypertension. We used a range of computational tools and evolutionary conservation analysis to build and validate a three-dimensional model of NHA2 based on the crystal structure of a distantly related bacterial transporter, NhaA. The model guided mutagenic evaluation of transport function, ion selectivity, and pH dependence of NHA2 by phenotype screening in yeast. We describe a cluster of essential, highly conserved titratable residues located in an assembly region made of two discontinuous helices of inverted topology, each interrupted by an extended chain. Whereas in NhaA, oppositely charged residues compensate for partial dipoles generated within this assembly, in NHA2, polar but uncharged residues suffice. Our findings led to a model for transport mechanism that was compared to the well-known electroneutral NHE1 and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.0Comments 27Citations