title: Functional analysis of the Na+,K+/H+ antiporter PeNHX3 from the tree halophyte Populus euphratica in yeast by model-guided mutagenesis.
abstract: Na+,K+/H+ antiporters are H+-coupled cotransporters that are crucial for cellular homeostasis. Populus euphratica, a well-known tree halophyte, contains six Na+/H+ antiporter genes (PeNHX1-6) 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+/H+ antiporter from the Escherichia coli (EcNhaA) as a template, we built the three-dimensional structure of PeNHX3 from P. euphratica. 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+, K+ and Li+ transport in a yeast growth assay. Domain-switch analysis shows that TM11 is crucial to Li+ transport. The novel features of PeNHX3 in ion binding and translocation are discussed.
authors.affiliation: MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, Gansu, China.
authors.forename: Liguang
authors.initials: L
authors.lastname: Wang
authors.affiliation: MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, Gansu, China.
authors.forename: Xueying
authors.initials: X
authors.lastname: Feng
authors.affiliation: MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, Gansu, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, Gansu, China.
authors.forename: Lidong
authors.initials: L
authors.lastname: Wang
authors.affiliation: MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, Gansu, China.
authors.forename: Lizhe
authors.initials: L
authors.lastname: An
authors.affiliation: MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, Gansu, China.
authors.forename: Quan-Sheng
authors.initials: QS
authors.lastname: Qiu
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001692:Biological Transport; D017124:Conserved Sequence; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008094:Lithium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010940:Plant Proteins / Q000737:chemistry; D032107:Populus / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D055051:Salt-Tolerant Plants / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014197:Trees / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D010940:Plant Proteins; D017923:Sodium-Hydrogen Exchangers; D008094:Lithium; D012964:Sodium; D011188:Potassium
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