Promiscuous Binding in a Selective Protein: The Bacterial Na+/H+ Antiporter

The ability to discriminate between highly similar substrates is one of the remarkable properties of enzymes. For example, transporters and channels that selectively distinguish between various solutes enable living organisms to maintain and control their internal environment in the face of a constantly changing surrounding. Herein, we examine in detail the selectivity properties of one of the most important salt transporters: the bacterial Na/H antiporter. Selectivity can be achieved at either the substrate binding step or in subsequent antiporting. Surprisingly, using both computational and experimental analyses synergistically, we show that binding per se is not a sufficient determinant of selectively. All alkali ions from Li to Cs were able to competitively bind the antiporter's binding site, whether the protein was capable of pumping them or not. Hence, we propose that NhaA's binding site is relatively promiscuous and that the selectivity is determined at a later stage of the transport cycle

Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights

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

Mechanism of Na+/H+ exchange by Escherichia coli NhaA in reconstituted proteoliposomes

AbstractPurified NhaA, a Na+/H+ antiporter from Escherichia coli, reconstituted into proteoliposomes was used to study partial reactions catalyzed by this protein. Homologous Na+/Na+ exchange as well as Na+/Li+ exchange via NhaA were detected by monitoring the effects of external Li+ and Na+ ions on the ΔpH-driven sodium uptake into NH4 Cl-loaded vesicles. Furthermore, a sodium counterflow reaction was demonstrated in proteoliposomes preloaded with non-radioactive Na+ and placed into the experimental buffer containing low amounts of 22Na+ under experimental conditions when both components of protonmotive force generated by the antiporter. ΔΨ and ΔpH, were dissipated by corresponding ionophores. The apparent Km for sodium counterflow is 1.1 mM, and Vmax is 80 μmol/minmg of protein. External Na+ accelerates the downhill efflux of 22Na+ suggesting that the translocation of the Na+loaded form of the carrier is faster than the rest of the catalytic cycle

Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in <em>Escherichia coli</em>

BACKGROUND: In neutralophilic bacteria, monovalent metal cation/H(+) antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis. RESULTS: Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na(+), K(+), Rb(+) or Li(+) for H(+). The K(+)/H(+) antiport reaction had a pH optimum at 9.0, whereas the Na(+)/H(+) exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress. CONCLUSIONS: Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli