title: Crystal structure of the plant dual-affinity nitrate transporter NRT1.1.
abstract: Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter that can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr 101; however, how this post-translational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr 101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 dimerizes in the cell membrane and that the phosphomimetic mutation of Thr 101 converts the protein into a monophasic high-affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.
authors.affiliation: Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, Washington 98195, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Bankston
authors.affiliation: 1] Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195, USA [2] Department of Structural Biology, Genentech Inc., South San Francisco, California 94080, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Payandeh
authors.affiliation: Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Hinds
authors.affiliation: Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, Washington 98195, USA.
authors.forename: William N
authors.initials: WN
authors.lastname: Zagotta
authors.affiliation: 1] Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195, USA [2] Howard Hughes Medical Institute, Box 357280, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Zheng
mesh_terms: D000595:Amino Acid Sequence; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D000091285:Nitrate Transporters; D009566:Nitrates / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011522:Protons; D013329:Structure-Activity Relationship
chemical_list: D027321:Anion Transport Proteins; C101492:NRT1.1 protein, Arabidopsis; D000091285:Nitrate Transporters; D009566:Nitrates; D010940:Plant Proteins; D011522:Protons; D010769:Phosphothreonine