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Nat Commun
2019 Jan 24;101:407. doi: 10.1038/s41467-018-08176-9.
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Crystal structure of the plant symporter STP10 illuminates sugar uptake mechanism in monosaccharide transporter superfamily.
Paulsen PA, Custódio TF, Pedersen BP.
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Plants are dependent on controlled sugar uptake for correct organ development and sugar storage, and apoplastic sugar depletion is a defense strategy against microbial infections like rust and mildew. Uptake of glucose and other monosaccharides is mediated by Sugar Transport Proteins, proton-coupled symporters from the Monosaccharide Transporter (MST) superfamily. We present the 2.4 Å structure of Arabidopsis thaliana high affinity sugar transport protein, STP10, with glucose bound. The structure explains high affinity sugar recognition and suggests a proton donor/acceptor pair that links sugar transport to proton translocation. It contains a Lid domain, conserved in all STPs, that locks the mobile transmembrane domains through a disulfide bridge, and creates a protected environment which allows efficient coupling of the proton gradient to drive sugar uptake. The STP10 structure illuminates fundamental principles of sugar transport in the MST superfamily with implications for both plant antimicrobial defense, organ development and sugar storage.
Fig. 1. Structure of the high affinity Sugar Transport Protein STP10. a The structure represents an outward facing occluded state of the sugar transporter in complex with glucose. Glucose (shown as spheres) is buried in the membrane at the interface between the N domain (blue) and C domain (green). Selected residues are shown as sticks. Black bars depict the approximate location of the membrane. b The glucose binding site towards the C domain. Yellow dashes indicate hydrogen bonds (2.6–3.6 Å distances) to glucose. The omit mFobs-DFcalc density for glucose is contoured in gold (5σ). c Same as panel b for the glucose binding site towards the N domain
Fig. 2. Functional characterization of STP10. a Glucose access from the extracellular side is blocked by the Lid domain covalently linked to the C domain. b Michaelis-Menten fit to glucose titration of STP10 using a Xenopus oocyte uptake assay at pH 5.0. c Binding affinity between glucose and STP10 by Isothermal titration calorimetry at pH 5.5. d Substrate specificity determined by competition in a yeast uptake assay at pH 5.0. *P < = 0.05; **P < = 0.01; and ***P < = 0.001 by Student’s t test. Data for all assays are mean ± SD of three or more replicate experiments
Fig. 3. The Lid domain and its effect on transport. a Electrostatic surface representation showing the negative cavity that connects the proton donor/acceptor site with the glucose binding site. A cluster of aromatic residues on the Lid domain isolate the proton site from the extracellular side. b Conservation of the aromatic residues of the Lid domain and residues of the proton donor/acceptor site and M1b. Both structure and sequence is colored according to sequence conservation between 1336 unique STPs (35–95% seq. ID) found across plant species. c Michaelis-Menten fit to glucose titration of STP10 mutant L43A at pH 4.0. d Glucose uptake rate as determined by a yeast uptake assay at different pH for WT STP10 and mutants C77A and C449A. Data for all assays are mean ± SD of three or more replicate experiments
Fig. 4. Proposed mechanism of glucose coupling to proton donor/acceptor site. In the outward open conformation (left), protons and glucose enter the central binding sites through small rearrangements of the N domain and the Lid domain that is covalently linked to the C domain through Cys77-Cys449. Protonation of Asp42 leads to its repulsion away from Arg142 and pushes the flexible M1b towards the glucose binding site, giving preference to high affinity glucose binding through Phe39 and Leu43 (right, observed structure). The aromatic cluster of the lid helps to isolate the proton donor/acceptor pair and maintain pKa values of Asp42 conductive to transport at a broad range of pH values
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