Article

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

Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
Proteins Structure Function and Bioinformatics (Impact Factor: 2.92). 08/2009; 76(3):548-59. DOI: 10.1002/prot.22368
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
55 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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. Na(+)/H(+) antiporters play primary role in pH homeostatic mechanisms both in prokaryotes and eukaryotes. Regulation by pH is a property common to these antiporters. They are equipped with a pH sensor to perceive the pH signal and a pH transducer to transduce the signal into a change in activity. Determining the crystal structure of NhaA, the Na(+)/H(+) antiporter of Escherichia coli have provided the basis for understanding in a realistic rational way the unique regulation of an antiporter by pH and the mechanism of the antiport activity. The physical separation between the pH sensor/transducer and the active site revealed by the structure entailed long-range pH-induced conformational changes for NhaA pH activation. As yet, it is not possible to decide whether the amino acid participating in the pH sensor and the pH transducer overlap or are separated. The pH sensor/transducer is not a single amino acid but rather a cluster of electrostatically interacting residues. Thus, integrating structural, computational, and experimental approaches are essential to reveal how the pH signal is perceived and transduced to activate the pH regulated protein. © 2011 American Physiological Society. Compr Physiol 1:1711-1719, 2011.
    10/2011; 1(4):1711-9. DOI:10.1002/cphy.c100078
  • [Show abstract] [Hide abstract]
    ABSTRACT: The crystal structure of down-regulated NhaA crystallized at acidic pH 4 [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. Extended chains interrupting helices appear now a common feature for ion binding in transporters. However the NhaA fold is unique and shared by ASBTNM [30] and NapA [29]. Computation [13], electrophysiology [69] combined with biochemistry [33,47] have provided intriguing models for the mechanism of NhaA. However, the conformational changes and the residues involved have not yet been fully identified. Another issue which is still enigma is how energy is transduced "in this 'nano-machine."' We expect that an integrative approach will reveal the residues that are crucial for NhaA activity and regulation, as well as elucidate the pHand ligand-induced conformational changes and their dynamics. Ultimately, integrative results will shed light on the mechanism of activity and pH regulation of NhaA, a prototype of the CPA2 family of transporters.
    Biochimica et Biophysica Acta 12/2013; DOI:10.1016/j.bbabio.2013.12.007 · 4.66 Impact Factor
  • [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.
    The Journal of General Physiology 12/2014; 144(6):529-44. DOI:10.1085/jgp.201411219 · 4.57 Impact Factor