Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs.
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.
School of Biology, University of St Andrews, St Andrews, Fife KY16 9TH, UK. su@st-and.ac.uk
Shiela E
SE
Unkles
Eugenia
E
Karabika
Vicki F
VF
Symington
Jennifer L
JL
Cecile
Duncan A
DA
Rouch
Naureen
N
Akhtar
Brett A
BA
Cromer
James R
JR
Kinghorn
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry; D001233:Aspergillus nidulans / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D001665:Binding Sites; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000091285:Nitrate Transporters; D009566:Nitrates / Q000378:metabolism; D010641:Phenotype; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D027321:Anion Transport Proteins; D005656:Fungal Proteins; D050505:Mutant Proteins; D000091285:Nitrate Transporters; D009566:Nitrates; C444100:NrtA protein, Aspergillus nidulans; D001216:Asparagine; D000409:Alanine; D005998:Glycine