Alanine Scanning Mutagenesis Of A High-Affinity Nitrate Transporter Highlights The Requirement For Glycine And Asparagine Residues In The Two Nitrate Signature Motifs

The high-affinity Major Facilitator Superfamily (MFS) nitrate transporter protein NrtA has proved thus far to be intractable to crystallographic procedures. Instead, structural models were developed and are presented here based on the crystal structures of the MFS members phosphate/glycerol-3-phosphate antiporter, GlpT and fucose/proton symporter, FucP. Alanine-scanning substitution of residues within and around the two nitrate signatures (NS1 and NS2) of NrtA have been analysed and show that residues within NS2 (TM11) are more tolerant of substitution by alanine than those in NS1 (TM5). In models of NrtA, asparagine residues N168 in NS1 and N459 in NS2 are positioned approximately midway between the outside and the inside faces of the protein at the central pivot point of the protein. N168 and N459 are in close proximity to substrate-binding arginine residues R368 and R87, respectively, which lie offset from the pivot point towards the cytoplasmic face. The N168/R368 and N459/R87 residue pairs are relatively widely separated on opposite sides of the likely substrate translocation pore. Results demonstrate the critical structural contribution of several glycine residues in each NS. These glycines are at sites of close helix packing and are likely to be important for protein folding and flexibility of TMs 5 and 11 as well as positioning of N168 and N459 correctly to allow proximity of the N168/R368 and N459/R87 residue pairs. Given the relative locations of N168/R368 and N459/R87 residue pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed