Structural and functional analysis of transmembrane segment IV of the salt tolerance protein Sod2

J Biol Chem. 2013 Aug 23;288(34):24609-24. doi: 10.1074/jbc.M113.483065. Epub 2013 Jul 8.

Abstract

Sod2 is the plasma membrane Na(+)/H(+) exchanger of the fission yeast Schizosaccharomyces pombe. It provides salt tolerance by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acid residues of transmembrane segment IV (TM IV) ((126)FPQINFLGSLLIAGCITSTDPVLSALI(152)) in activity by using alanine scanning mutagenesis and examining salt tolerance in sod2-deficient S. pombe. Two amino acids were critical for function. Mutations T144A and V147A resulted in defective proteins that did not confer salt tolerance when reintroduced into S. pombe. Sod2 protein with other alanine mutations in TM IV had little or no effect. T144D and T144K mutant proteins were inactive; however, a T144S protein was functional and provided lithium, but not sodium, tolerance and transport. Analysis of sensitivity to trypsin indicated that the mutations caused a conformational change in the Sod2 protein. We expressed and purified TM IV (amino acids 125-154). NMR analysis yielded a model with two helical regions (amino acids 128-142 and 147-154) separated by an unwound region (amino acids 143-146). Molecular modeling of the entire Sod2 protein suggested that TM IV has a structure similar to that deduced by NMR analysis and an overall structure similar to that of Escherichia coli NhaA. TM IV of Sod2 has similarities to TM V of the Zygosaccharomyces rouxii Na(+)/H(+) exchanger and TM VI of isoform 1 of mammalian Na(+)/H(+) exchanger. TM IV of Sod2 is critical to transport and may be involved in cation binding or conformational changes of the protein.

Keywords: Alanine Scanning Mutagenesis; Cation Coordination; Membrane Function; Membrane Proteins; Na+/H+ Exchanger; Nuclear Magnetic Resonance; Salt Tolerance; Sodium Transport; Sodium/Proton Exchange.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Mutation, Missense
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Schizosaccharomyces / chemistry
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / chemistry
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Sodium-Hydrogen Exchangers / chemistry
  • Sodium-Hydrogen Exchangers / genetics
  • Sodium-Hydrogen Exchangers / metabolism*
  • Structural Homology, Protein

Substances

  • Escherichia coli Proteins
  • Fungal Proteins
  • NhaA protein, E coli
  • SOD2 protein, Zygosaccharomyces rouxii
  • Schizosaccharomyces pombe Proteins
  • Sod2 protein, S pombe
  • Sodium-Hydrogen Exchangers

Associated data

  • PDB/2M7X