Abstract
Sugars are essential sources of energy and carbon and also function as key signalling molecules in plants. Sugar transport proteins (STP) are proton-coupled symporters responsible for uptake of glucose from the apoplast into plant cells. They are integral to organ development in symplastically isolated tissues such as seed, pollen and fruit. Additionally, STPs play a vital role in plant responses to stressors such as dehydration and prevalent fungal infections like rust and mildew. Here we present a structure of Arabidopsis thaliana STP10 in the inward-open conformation at 2.6 Å resolution and a structure of the outward-occluded conformation at improved 1.8 Å resolution, both with glucose and protons bound. The two structures describe key states in the STP transport cycle. Together with molecular dynamics simulations that establish protonation states and biochemical analysis, they pinpoint structural elements, conserved in all STPs, that clarify the basis of proton-to-glucose coupling. These results advance our understanding of monosaccharide uptake, which is essential for plant organ development, and set the stage for bioengineering strategies in crops.
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Data availability
Coordinates and structure factors from this study have been deposited in the Protein Data Bank with the accession numbers 7AAQ (outward) and 7AAR (inward). Previously published coordinates used for molecular replacement are available at the Protein Data Bank (accession number 6H7D). Source data are provided with this paper.
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Acknowledgements
We acknowledge beamlines I24 and I04 at the Diamond Light Source and beamline BioMAX at the MAX IV Laboratory, where X-ray data were collected, as well as DESY-PETRA III for crystal screening. This work was supported by funding from the Danish Council for Independent Research (grant agreement no. DFF-4002-00052), the Carlsberg Foundation (CF17-0180) and an AIAS fellowship to B.P.P. Novo Nordisk Foundation (NNF18OC0052988), the Villum Foundation (project number 34326) and the Independent Research Fund Denmark, Natural Sciences (7014-00192B) supported J.C.F.-C. Computations were performed at the Grendel-S cluster of the Centre for Scientific Computing Aarhus (CSC-AA) and made possible by a grant from the Novo Nordisk Foundation (NNF18OC0032608).
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L.B. carried out crystallization experiments, processed data and performed biochemical characterization. P.A.P. carried out crystallization experiments and processed data. J.C.F.-C. performed MD simulations. B.S. supervised the MD simulations. B.P.P. supervised the project. L.B. and B.P.P. wrote the paper. All authors commented on the paper.
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Extended data
Extended Data Fig. 1 Crystals and components of the asymmetric unit.
a) SDS-PAGE gel of STP10 protein and polarized light photo of STP10 wild type crystals and light photo of STP10 E162Q D344N crystals. Pictures are representative of 50+ purification and crystallization trials that gave comparable results. b) Asymmetric unit and crystal packing of STP10 wild type. The unit cell is viewed perpendicular to the ab-plane, and the a and b axis highlighted in red. The asymmetric unit contains one molecule of STP10, as highlighted in darker colors. The packing is an example of type I packing normally obtained by LCP crystallography with the transmembrane regions packing in a lipid bilayer and a relatively low solvent content (58%). c) The backbone of STP10 outward occluded structure and inward open structure colored by the atomic displacement factor (B-factor) with a rainbow gradient from low/blue to high/red. There is a disordered loop between M9 and M10 with a notably higher B-factor than the rest of the model in the outward structure and a disordered part of the Lid domain with notably higher B-factor than the rest of the model in the inward open structure. d) Asymmetric unit and crystal packing of STP10 E162Q D344N. The unit cell is viewed perpendicular to the bc-plane, and the b and c axis highlighted in red. The asymmetric unit contains one molecule of STP10, as highlighted in darker colors.
Extended Data Fig. 2 Electron density for the STP10 outward occluded structure and the STP10 inward open structure.
a) Weighted 2FoFc density at 1.5 sigma of the asymmetric unit of 1.8 Å resolution STP10 outward occluded structure with the final model overlaid. b) Weighted 2FoFc density at 1.0 sigma of the asymmetric unit of 2.6 Å resolution STP10 inward open structure with the final model overlaid.
Extended Data Fig. 3 Electron density for selected components of STP10 structures.
a) Backbone representation of the STP10 outward structure with all heterologous molecules found in the density highlighted. Besides STP10 the model contains 1 glucose, 2 acetate, 1 PEG 458, 4 monoolein molecules and 157 waters. The four inserts highlight the quality of the electron density displayed by the weighted 2FoFc density at 1.5 sigma for the PEG and acetate, glucose, intracellular cysteines and of the monoolein, which was weaker and is clearer at lower sigma levels than 1.5. b) Backbone representation of the STP10 inward structure with all the heterologous molecules found in the density highlighted. Besides STP10 the model contains 1 glucose, 1 chloride ion, 2 OGNG and 9 waters. The four inserts highlight the quality of the electron density displayed by the weighted 2FoFc density at 1.0 sigma for the two OGNG, glucose and the disulfide bridge.
Extended Data Fig. 4 The intracellular gate in the two STP10 structures.
a) View of the STP10 outward occluded structure perpendicular to the membrane with the three key interdomain salt bridge networks highlighted in colored squares. In particular constituted by the double salt bridge from D344(M8) to the main chain nitrogen of Gly170(M5) and Ala171(M5) (network 1) and the double salt bridge from Glu162 (M4) to Arg422(M11), Thr226 (IC1) and R111(M3) (network 2) as well as and from Arg169(M5) to E415 (M10), the main chain carbonyls of Thr477 (IC5) and Val480 (IC5) (network 3). These regions are perfectly conserved in all STPs and in several bacterial symporters, and have also been observed in human sugar facilitators. b) Close-up view of the N domain and C domain at the cytosolic side in the STP10 inward open structure. In the inward open conformation, interactions between the ICH domain and the transmembrane N and C domains are maintained. Interactions between ICH and the two transmembrane domain residues are highlighted by yellow dashes. The mutant residues Q162 and N344 that broke the stabilizing networks are highlighted in red. The positions of the SP motifs are highlighted (dotted eclipses).
Extended Data Fig. 5 Functional characterization of STP10 mutants.
a) Uptake of glucose into EBY.VW4000 yeast strain expressing STP10 (black circles), various mutants (squares and triangles) or empty plasmid (empty circles) per OD600 of cells at an initial outside concentration of 100 μM glucose at pH 5.0. b) STP10 inhibition determined by EBY.VW4000 competition assay at pH 5.0. While cold glucose competes with labeled glucose uptake, neither Forskolin or N-Acetylglucosamin affects glucose uptake. All competition was done at 50x molar excess. ****, P < = 0.0001 by Student’s t test (two-tailed). Data are presented as mean ± SD (n = 4 independent experiments). c–f) Michaelis-Menten fit to glucose titration of STP10 mutants at pH 5.0. Data are presented as mean ± SD (n = 3 independent experiments).
Extended Data Fig. 6 Molecular dynamics simulations of the glucose binding site of STP10 inward open states.
a) R.m.s.d. backbone plot of ten independent repeats for the outward and inward state with Asp42 either neutral or charged. b) R.m.s.d. ligand plot of the charged inward open state simulations. The glucose leaves the inward state in 4 of 10 independent repeats with Asp42 charged. c) R.m.s.d. ligand plot of the neutral inward open state simulations. The glucose leaves the inward state in 6 of 10 independent repeats with Asp42 neutral. Repeats are represented by different color traces same as for Fig. 3d. The ligand RMSD is calculated after aligning the protein structure to the initial model.
Extended Data Fig. 7 Continuum electrostatics and empirical pKa calculations for the two structures.
a) pK1/2 for titratable residues in STP10 for the outward crystal structure with different dielectric constant values for the protein (4, 6 and 10). Glu162 has pK1/2 values < -4 for all three dielectric constant values. His492 has pK1/2 < -4 for a dielectric constant of 4. Predicted pKa using Propka3.0, an empirical method. See subsection Protonation states assignment in Methods for calculation details. b) pK1/2 for titratable residues in STP10 for the inward crystal structure with different dielectric constant values for the protein (4, 6 and 10). Predicted \pKa using the empirical method Propka3.0. See subsection Protonation states assignment in Methods for calculation details.
Extended Data Fig. 8 Multiple sequence alignment of the A. thaliana Sugar Transport Family STP9, STP10, STP11 with other plant STPs included.
Alignment between A. thaliana STP9 (accession number Q9SX48), A. thaliana STP10 (accession number Q9LT15), A. thaliana STP11 (accession number Q9FMX3), Cucumis melo cmSTP10-like (accession numberA0A5A7SS92), Theobroma cacao tcSTP (accession number A0A061E224), Populus trichocarpa ptSTP (accession number B9H5Q5), Manihot esculent meSTP (accession number A0A2C9V070), Cucumis sativus csSTP (acces-sion number A0A0A0LHS6), Brassica pekinensis bpSTP (accession num-ber M4FAX8), Capsella rubella crSTP (accession number R0I4Q9), Glycine hispida ghSTP (accession number I1LF83) and Mucuna pruriens mpSTP10 (accession number A0A371FNF1). Conserved residues are highlighted with gray-scale, where black is perfectly conserved. Colored tubes represent α-helices found in the N domain (blue), Lid domain (orange), ICH domain(pale yellow) and C domain (green). Key residues are numbered above the α-helix markings. Residues highlighted in red participate in sugar binding. The proton donor/acceptor pair is highlighted in green. The cysteines forming the disulfide bridge between Lid domain and C domain as well as the cysteines at the intracellular interface are highlighted in yellow. The tyrosines involved in exofacial gating are highlighted in magenta. Conserved motifs are highlighted in light blue.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2.
Supplementary Video 1
Steps in the morph were calculated using Morphit Pro with default settings (http://morphit-pro.cmp.uea.ac.uk/MorphItPro/).
Source data
Source Data Fig. 2
Uptake assay, raw counts per minute.
Source Data Fig. 3
Uptake assay, raw counts per minute.
Source Data Fig. 4
Uptake assay, raw counts per minute.
Source Data Extended Data Fig. 5
Uptake assay, raw counts per minute.
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Bavnhøj, L., Paulsen, P.A., Flores-Canales, J.C. et al. Molecular mechanism of sugar transport in plants unveiled by structures of glucose/H+ symporter STP10. Nat. Plants 7, 1409–1419 (2021). https://doi.org/10.1038/s41477-021-00992-0
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DOI: https://doi.org/10.1038/s41477-021-00992-0
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