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

Unkles, S, Karabika, E, Symington, V, Cecile, J, Rouch, D, Akhtar, N, Cromer, B and Kinghorn, J 2012, 'Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs', Biochemical Journal, vol. 447, no. 1, pp. 35-42.


Document type: Journal Article
Collection: Journal Articles

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Title Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs
Author(s) Unkles, S
Karabika, E
Symington, V
Cecile, J
Rouch, D
Akhtar, N
Cromer, B
Kinghorn, J
Year 2012
Journal name Biochemical Journal
Volume number 447
Issue number 1
Start page 35
End page 42
Total pages 8
Publisher Portland Press Ltd
Abstract Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 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 Arg 87 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 /Arg 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
Subject Receptors and Membrane Biology
Keyword(s) alanine scanning
helix packing
major facilitator superfamily (MFS)
nitrate transporter
structural model
DOI - identifier 10.1042/BJ20120631
Copyright notice © The Authors Journal compilation © 2012 Biochemical Society
ISSN 0264-6021
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