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Structures of bacterial homologues of SWEET transporters in two distinct conformations

Nature volume 515, pages 448–452 (2014)Cite this article

Subjects

Abstract

SWEETs and their prokaryotic homologues are monosaccharide and disaccharide transporters that are present from Archaea to plants and humans1,2,3. SWEETs play crucial roles in cellular sugar efflux processes: that is, in phloem loading4, pollen nutrition5 and nectar secretion6. Their bacterial homologues, which are called SemiSWEETs, are among the smallest known transporters1,3. Here we show that SemiSWEET molecules, which consist of a triple-helix bundle, form symmetrical, parallel dimers, thereby generating the translocation pathway. Two SemiSWEET isoforms were crystallized, one in an apparently open state and one in an occluded state, indicating that SemiSWEETs and SWEETs are transporters that undergo rocking-type movements during the transport cycle. The topology of the triple-helix bundle is similar yet distinct to that of the basic building block of animal and plant major facilitator superfamily (MFS) transporters (for example, GLUTs and SUTs). This finding indicates two possibilities: that SWEETs and MFS transporters evolved from an ancestral triple-helix bundle or that the triple-helix bundle represents convergent evolution. In SemiSWEETs and SWEETs, two triple-helix bundles are arranged in a parallel configuration to produce the 6- and 6 + 1-transmembrane-helix pores, respectively. In the 12-transmembrane-helix MFS transporters, four triple-helix bundles are arranged into an alternating antiparallel configuration, resulting in a much larger 2 × 2 triple-helix bundle forming the pore. Given the similarity of SemiSWEETs and SWEETs to PQ-loop amino acid transporters and to mitochondrial pyruvate carriers (MPCs), the structures characterized here may also be relevant to other transporters in the MtN3 clan7,8,9. The insight gained from the structures of these transporters and from the analysis of mutations of conserved residues will improve the understanding of the transport mechanism, as well as allow comparative studies of the different superfamilies involved in sugar transport and the evolution of transporters in general.

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Figure 1: Structure of Vibrio sp. SemiSWEET.
Figure 2: Structure of L. biflexa SemiSWEET.
Figure 3: Glucose transport by L. biflexa SemiSWEET and A. thaliana SWEET1.
Figure 4: Alternating access by the rocking-type movement of two protomers.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Crystallographic coordinates and structure factors of L. biflexa SemiSWEET and Vibrio sp. SemiSWEET have been deposited in the Protein Data Bank database under accession numbers 4QNC and 4QND, respectively.

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Acknowledgements

We thank the staff at beamlines 23ID-B and 23ID-D (APS, Argonne National Laboratory) and S. Russi and the staff at beamlines 11-1 and 12-2 (SSRL, SLAC National Laboratory) for assistance at the synchrotrons. We thank the Kobilka laboratory for help and advice on the LCP. This work was made possible by support from Stanford University and the Harold and Leila Y. Mathers Charitable Foundation to L.F. and from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences at the US Department of Energy (DOE) under grant number DE-FG02-04ER15542 to W.B.F. Part of this work is based upon research conducted at the APS on the Northeastern Collaborative Access Team beamlines, which are supported by a grant from the National Institute of General Medical Sciences (P41 GM103403) from the National Institutes of Health. Use of the APS, an Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory, was supported by the DOE under contract number DE-AC02-06CH11357.

Author information

Author notes

  1. Yan Xu, Yuyong Tao and Lily S. Cheung: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, 279 Campus Drive, Stanford University School of Medicine, Stanford, 94305, California, USA

    Yan Xu, Yuyong Tao, Chao Fan & Liang Feng

  2. Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, California 94305, USA,

    Lily S. Cheung, Li-Qing Chen & Wolf B. Frommer

  3. Department of Biology, Stanford University, Stanford, 94305, California, USA

    Sophia Xu & Wolf B. Frommer

  4. NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, USA,

    Kay Perry

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Contributions

Y.X., W.B.F. and L.F. conceived and designed experiments. Y.X. and Y.T. performed expression, purification, crystallization, data collection and crystallography. C.F. performed functional experiments. S.X. performed biochemical characterization. L.S.C. and L.-Q.C. performed alignments and functional experiments. K.P. performed data collection and assisted crystallography. L.F. contributed to crystallization, data collection and crystallography. Y.X., Y.T., L.S.C., C.F., L.-Q.C., W.B.F. and L.F. analysed the data. L.F. and W.B.F. wrote the manuscript.

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Correspondence to Liang Feng.

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Extended data figures and tables

Extended Data Figure 1 Sequence alignment.

The secondary structure is shown above the alignment. Protein sequences were aligned using ClustalW and manually adjusted. The N-terminal part (first THB) and C-terminal part (second THB) of A. thaliana SWEET1 were separately used for alignment and are labelled A. thaliana SWEET1-N and A. thaliana SWEET1-C, respectively.

Extended Data Figure 2 Vibrio sp. SemiSWEET dimer in the crystal.

Crystal packing in the lipid bilayer environment shows the formation of the dimer. One protomer is shown in green, and the other is shown in purple.

Extended Data Figure 3 Subunit stoichiometry of SemiSWEET.

a, Purified Vibrio sp. SemiSWEET (predicted molecular weight, 11.1 kDa) remains as a dimer after SDS–PAGE. b, Crosslinking of purified B. japonicum SemiSWEET (theoretical molecular weight, 10.1 kDa) in detergent solution. Increasing amounts of disuccinimidyl suberate (DSS; 0.02 mM, 0.2 mM and 2 mM) were used to crosslink B. japonicum SemiSWEET. The protein was analysed by SDS–PAGE and stained with Coomassie blue.

Extended Data Figure 4 Crystal packing of L. biflexa SemiSWEET.

The crystal lattice structure of L. biflexa SemiSWEET shows dimer formation in the membrane. One protomer is shown in red, and the other is shown in blue.

Extended Data Figure 5 Conserved tryptophan and asparagine residues in A. thaliana SWEET1 are required for transport activity.

a, Functional analysis of A. thaliana SWEET1–GFP (green fluorescent protein) fusion transport activity in the EBY4000 yeast strain. The point mutations W56A, N73A, W176A and N192A failed to complement the growth defect of EBY4000 in synthetic medium supplemented with 2% glucose as the only carbon source. Growth was unaffected in control medium containing 2% maltose. An empty vector and A. thaliana SWEET1–GFP were used as the negative and positive controls, respectively. b–g, Membrane localization of the wild-type and mutant A. thaliana SWEET1 was confirmed by imaging of GFP. The empty vector negative control (b) shows the cytoplasmic localization of GFP, while the wild-type and W56A, N73A, W176A and N192A mutants (c–g) show membrane localization, suggesting that the phenotype of the mutated transporters is not due to mislocalization.

Extended Data Figure 6 Transmembrane-helix arrangements in SWEET, MFS and G-protein-coupled receptor (GPCR) proteins.

Ribbon representations of a three-transmembrane-helix unit in a SWEET (left), a GPCR (centre) and an MFS (right) are shown (top), with their respective topologies (bottom). The same colour scheme is used for all three proteins.

Extended Data Figure 7 Proposed transport model and pathway for building SWEET and MFS transporters.

a, Alternating access model for SemiSWEET transport. One protomer is shown in purple, and the other is shown in green. The putative substrate is shown as an orange hexagon. b, Proposed pathway for constructing SemiSWEET, SWEET or MFS transporters from the primitive THB. The same colour scheme is used for all THBs. The position of TM3 relative to TM1 and TM2 of the THB in SWEET or MFS is illustrated by coloured circles, viewed from the N terminus. Note that the THB in SWEET and the THB in MFS are roughly mirror images of each other.

Extended Data Table 1 Data collection and refinement statistics
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Xu, Y., Tao, Y., Cheung, L. et al. Structures of bacterial homologues of SWEET transporters in two distinct conformations. Nature 515, 448–452 (2014). https://doi.org/10.1038/nature13670

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