Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs

Shiela E. Unkles*, Eugenia Karabika, Vicki F. Symington, Jennifer L. Cecile, Duncan A. Rouch, Naureen Akhtar, Brett A. Cromer, James R. Kinghorn

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and II termed NS (nitrate signature) I and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn(168) in NS1 and Asn(459) in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg(368) and Are respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn(168)/Arg(368) and Asn(459)/Arg(87) residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn(168)/Are(368) and Asn(459)/Arg(87) pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed.

Original languageEnglish
Pages (from-to)35-42
Number of pages8
JournalBiochemical Journal
Volume447
DOIs
Publication statusPublished - 1 Oct 2012

Keywords

  • alanine scanning
  • helix packing
  • major facilitator superfamily (MFS)
  • nitrate transporter
  • structural model
  • ESCHERICHIA-COLI
  • ASPERGILLUS-NIDULANS
  • OXALOBACTER-FORMIGENES
  • SUBSTRATE-BINDING
  • GLYCEROL-3-PHOSPHATE TRANSPORTER
  • CONFORMATIONAL-CHANGES
  • OXALATE TRANSPORTER
  • PROTEIN-STRUCTURE
  • MODEL
  • MECHANISM

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