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, which 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, Thr101. Yet how this posttranslational 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 Thr101 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 indeed dimerizes in the cell membrane and the phosphomimetic mutation of Thr101 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.