Functional Analysis of the Na<sup>+</sup>,K<sup>+</sup>/H<sup>+</sup> Antiporter PeNHX3 from the Tree Halophyte <i>Populus euphratica</i> in Yeast by Model-Guided Mutagenesis

<div><p>Na<sup>+</sup>,K<sup>+</sup>/H<sup>+</sup> antiporters are H<sup>+</sup>-coupled cotransporters that are crucial for cellular homeostasis. <i>Populus euphratica</i>, a well-known tree halophyte, contains six Na<sup>+</sup>/H<sup>+</sup> antiporter genes (<i>PeNHX1-6</i>) that have been shown to function in salt tolerance. However, the catalytic mechanisms governing their ion transport remain largely unknown. Using the crystal structure of the Na<sup>+</sup>/H<sup>+</sup> antiporter from the <i>Escherichia coli</i> (EcNhaA) as a template, we built the three-dimensional structure of PeNHX3 from <i>P. euphratica</i>. The PeNHX3 model displays the typical TM4-TM11 assembly that is critical for ion binding and translocation. The PeNHX3 structure follows the ‘positive-inside’ rule and exhibits a typical physicochemical property of the transporter proteins. Four conserved residues, including Tyr149, Asn187, Asp188, and Arg356, are indentified in the TM4-TM11 assembly region of PeNHX3. Mutagenesis analysis showed that these reserved residues were essential for the function of PeNHX3: Asn187 and Asp188 (forming a ND motif) controlled ion binding and translocation, and Tyr149 and Arg356 compensated helix dipoles in the TM4-TM11 assembly. PeNHX3 mediated Na<sup>+</sup>, K<sup>+</sup> and Li<sup>+</sup> transport in a yeast growth assay. Domain-switch analysis shows that TM11 is crucial to Li<sup>+</sup> transport. The novel features of PeNHX3 in ion binding and translocation are discussed.</p></div>