Functional analysis of the Na+,K+/H+ antiporter PeNHX3 from the tree halophyte Populus euphratica in yeast by model-guided mutagenesis

PLoS One. 2014 Aug 5;9(8):e104147. doi: 10.1371/journal.pone.0104147. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Biological Transport
  • Conserved Sequence
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Hydrophobic and Hydrophilic Interactions
  • Lithium / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis*
  • Plant Proteins / chemistry
  • Populus / metabolism*
  • Potassium / metabolism
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / metabolism*
  • Salt-Tolerant Plants / metabolism*
  • Sodium / metabolism
  • Sodium-Hydrogen Exchangers / chemistry
  • Sodium-Hydrogen Exchangers / metabolism*
  • Structure-Activity Relationship
  • Trees / metabolism*

Substances

  • Escherichia coli Proteins
  • NhaA protein, E coli
  • Plant Proteins
  • Sodium-Hydrogen Exchangers
  • Lithium
  • Sodium
  • Potassium

Grants and funding

This work was supported by the National Basic Research Program of China (973 project, 2013CB429904 to QSQ), the National Natural Science Foundation of China (NSFC) (31371438, 31070222 to QSQ), the Research Fund for the Doctoral Program of Higher Education of China (RFDP) (20130211110001 to QSQ), and the Fundamental Research Funds for the Central Universities (lzujbky-2013-k10). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.