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Crystal structure of the plant dual-affinity nitrate transporter NRT1.1

Nature volume 507, pages 73–77 (2014)Cite this article

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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.

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Figure 1: Crystal packing and overall structure of NRT1.1.
Figure 2: NRT1.1 dimer interface.
Figure 3: NRT1.1 dimerization controlled by Thr 101 phosphorylation.
Figure 4: Substrate binding and energy coupling in NRT1.1.
Figure 5: A dimerization switch model.

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Protein Data Bank

Data deposits

Structural coordinates and structural factors are deposited in the Protein Data Bank under accession number 4OH3.

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Acknowledgements

We thank the beamline staff of the Advanced Light Source at the University of California at Berkeley. We also thank members of the Zheng laboratory, Zagotta laboratory, Xu laboratory and H. Zheng for discussion and help. This work is supported by the Howard Hughes Medical Institute (N. Z.), National Institutes of Health (R01EY10329 to W.N.Z., NS074545 to J.R.B.) and the National Science Foundation (N.Z.).

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  1. Jian Payandeh

    Present address: Present address: Department of Structural Biology, Genentech Inc., South San Francisco, California 94080, USA.,

Authors and Affiliations

  1. Department of Pharmacology, Box 357280, University of Washington, Seattle, 98195, Washington, USA

    Ji Sun, Jian Payandeh, Thomas R. Hinds & Ning Zheng

  2. Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, 98195, Washington, USA

    John R. Bankston & William N. Zagotta

  3. Howard Hughes Medical Institute, Box 357280, University of Washington, Seattle, 98195, Washington, USA

    Ning Zheng

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Contributions

J.S. and N.Z. conceived and J.S. conducted the protein purification and crystallization experiments. J.P. provided experimental suggestions. J.S. and N.Z. determined and analysed the structures. J.S., T.R.H. and N.Z. conceived and J.S. and T.R.H. conducted SEC-LS-RI-UV experiments. J.S., J.R.B., W.N.Z. and N.Z. conceived and J.S. and J.R.B. conducted FRET experiments. J.S. and N.Z. conceived and J.S. conducted mutational and transporter assays. J.S. and N.Z wrote the manuscript with inputs from all authors.

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Correspondence to Ning Zheng.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequence alignment of plant NRT1.1 orthologues.

Alignment and secondary structure assignments of NRT1.1 orthologues from Arabidopsis thaliana (At), Brassica napus (Bn), Oryza sativa (Os), Sorghum bicolor (Sb), Populus trichocarpa (Pt), Vitis vinifera (Vv) and Zea mays (Zm). Strictly conserved residues are coloured in blue. Green dots indicate the EXXER motif. Orange empty squares indicate dimer interface residues. Red triangles indicate residues in the substrate-binding pocket. The red dot indicates the energy-coupling residue. Dashed lines represent the disordered region in the crystal structure.

Extended Data Figure 2 Sequence alignment of Arabidopsis NRT1 family members.

Alignment and secondary structure assignments of the NRT1 family members from Arabidopsis thaliana. Strictly conserved residues are coloured in blue. The two residues potentially important for substrate binding are indicated by a red triangle with green stroke. The energy coupling residue is indicated by a red dot. Dashed lines represent the disordered region in the crystal structure.

Extended Data Figure 3 Nitrate uptake and electron density map of NRT1.1.

a, Measurement of nitrate uptake by NRT1.1 was carried out in Xenopus oocytes in the presence of increasing concentrations of nitrate. A Q-test was used to identify statistical outliers in data. All data points are mean ± s.d. of one experiment in quintuplicates or sextuplicates. The data were fit with a two site nonlinear binding curve using Prism. b, Relative nitrate uptake activities of NRT1.1 mutants to the wild-type protein measured in the Xenopus oocyte-based assay in the presence of 10 mM nitrate. All results are the mean ± s.d. of one experiment in quintuplicates or sextuplicates. c, The overall 2Fo − Fc map of the NRT1.1 dimer contoured at 1.5σ. d, e, Two representative helixes and their 2Fo − Fc maps contoured at 1.5σ. f, An island of density from the 2Fo − Fc map contoured at 1.5σ is assigned to the head group of DDM bound between TMH5 and TMH8.

Extended Data Figure 4 Structural comparison of NRT1.1 and other MSF transporters.

a, Cutaway views of PepTst, GlpT and LacY showing their shared inward conformation as observed for NRT1.1. b, Overall structural comparison of NRT1.1 and PepTst with their N-terminal and C-terminal domains (NTD and CTD) coloured in pale green and cyan, respectively. Superposition of their NTDs and CTDs are shown separately. c, Comparisons between NRT1.1 and two bacterial NRT2 family nitrate transporters, NarK and NarU.

Extended Data Figure 5 Assessment of the oligomerization state of detergent-solubilized NRT1.1.

a, SEC-LS-RI-UV analysis of DDM-solubilized NRT1.1. Normalized light scattering signal from the 90° detector, ultraviolet absorption signal, and the refractive index signals are plotted in blue, green and red lines, respectively. The highest peak contains NRT1.1 bound to DDM. The calculated masses of the protein-detergent micelle complex (magenta), DDM micelle (cyan), and the NRT1.1 protein (black) are shown. The complex contains about 196 detergent molecules (molecular mass 510.62 Da) and 1 NRT1.1 molecule (67 kDa). The second peak belongs to the detergent micelle with a mass of 79 kDa or 155 detergent molecules. b, Crosslinking of the NRT1.1 T101D mutant protein with increasing concentrations of EGS. The protein was purified in the presence of 0.1% digitonin.

Extended Data Figure 6 Shape complementarity and conservation of the NRT1.1 dimer interface.

a, Two representative cross-section views of the NRT1.1 dimer interface that are parallel to the membrane. The top cross-section goes through the plane defined by Ala 110 and Val 229 in the two NRT1.1 protomers. The bottom cross-section goes through Ala 104 and Ala 237. b, Conservation surface mapping of NRT1.1 residues at the dimer interface among NRT1.1 orthologues (left) and among Arabidopsis NRT1 family members (right). A colour ramp (white, pale yellow, bright orange, to deep orange) is used to indicate the degree of conservation of surface residues. The arrow indicates the N-terminal segment.

Extended Data Figure 7 Spatial relationship between the N terminus of the two NRT1.1 protomers.

a, The N termini of the two NRT1.1 protomers in the crystal structure are about 42 Å apart and are shown in two orthogonal orientations. b, Spectral quantification of the FRET. Emission spectra measured from an oocyte expressing wild-type NRT1.1 (top left), NRT1.1(T101D) (top right), NRT1.1(T101A) (bottom left), and wild-type mCitrine–NRT1.1 and mCerulean–HCN2 (bottom right). The spectra are colour coded as follows: cyan, 458 nm excitation of oocytes expressing mCerulean constructs alone; black, 488 nm excitation of oocytes expressing both mCitrine and mCerulean constructs; red, 458 nm excitation of oocytes expressing both mCitrine and mCerulean constructs; green, subtracted spectrum (red minus cyan). The dashed line is the position of the peak of the fluorescence signal after excitation at 458 nm of the mCitrine–NRT1.1 only expressing oocytes (the position of the Azero or no FRET peak).

Extended Data Figure 8 Putative nitrate-binding site in the two NRT1.1 protomers.

a, b, Intracellular view of the substrate binding site in the two copies of NRT1.1 within the dimer. To compare the relative position of the substrate to its surrounding residues, distances (Å) between the nitrogen atom of the modelled nitrate and select amino acid atoms in its vicinity are shown with dashed lines and are indicated. Nitrate is shown in sticks with electron density contoured at 4σ from a Fo − Fc map calculated without the substrate. TMHs are numbered. c, d, Side view of the substrate binding site. e, f, A comparison of the putative substrate density between the NRT1.1 structures determined with a cryo-protectant solution containing 10 mM or 0 mM nitrate. g–i, Electrostatic potential surface of the NRT1.1 substrate pocket. The surface colours are clamped between red (−20 kT e−1) and blue (+20 kT e−1). Nitrate is shown as spheres. Two global views of the electrostatic potential surface of NRT1.1 are shown for comparison.

Extended Data Figure 9 The conserved cleft formed by the N-terminal segment of NRT1.1.

Overall and close-up views of the N-terminal segments of NRT1.1 within the dimer are shown in surface representation. The cleft-forming residues, which are strictly conserved in the NRT1.1 orthologues, are labelled and shown in stick representation.

Extended Data Table 1 Data collection, phasing and refinement statistics (SAD)
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Sun, J., Bankston, J., Payandeh, J. et al. Crystal structure of the plant dual-affinity nitrate transporter NRT1.1. Nature 507, 73–77 (2014). https://doi.org/10.1038/nature13074

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