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Structure and mechanism of the mammalian fructose transporter GLUT5

Nature volume 526, pages 397–401 (2015)Cite this article

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Abstract

The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a ‘gated-pore’ transport mechanism in such monosaccharide transporters.

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Figure 1: Structures of rat GLUT5 in the open outward-facing conformation and bovine GLUT5 in the open inward-facing conformation.
Figure 2: The fructose-binding site of GLUT5.
Figure 3: Inter-TM salt bridges form between bundle cytoplasmic ends in the outward-facing conformation.
Figure 4: Substrate-induced gates are predominantly formed by TM7 and TM10 in the C-terminal bundle.
Figure 5: Alternating-access transport mechanism in GLUT5.

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Primary accessions

Protein Data Bank

Data deposits

The coordinates and the structure factors for bovine and rat GLUT5 have been deposited in the Protein Data Bank under accessions 4YB9 and 4YBQ, respectively.

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Acknowledgements

We are grateful to D. Slotboom, A. Cameron and S. Newstead for discussions and comments, and J. Mansfield for assistance with large-scale yeast fermentations and H. Unno with rGLUT5 crystallization. Data were collected at the European Synchrotron Radiation Facility, Diamond Light Source, and SPring-8 (proposal numbers 2011A1393, 2011B1229, 2012A1184, 2012B1253, 2013A1241, 2013B1237, 2014A1348 and 2014B1407), with assistance from beamline scientists. This work was funded by the Knut and Alice Wallenberg Foundation (D.D), The Royal Society through the University Research Fellow scheme (D.D), the BBSRC (BB/G02325/1 to S.I.), the ERATO Human Receptor Crystallography Project of the Japan Science and Technology Agency (JST) (S.I.), by the Research Acceleration Program of the JST (S.I.), by the Targeted Proteins Research Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (S.I.), and by Grants-in-Aids for Scientific Research from the MEXT (No. 22570114 to N.N.), and by the Platform for Drug Discovery, Informatics, and Structural Life Science from the MEXT (T.K.). The authors are grateful for the use of the Membrane Protein Laboratory funded by the Wellcome Trust (grant 062164/Z/00/Z) at the Diamond Light Source Limited, and The Centre for Biomembrane Research (CBR), supported by the Swedish Foundation for Strategic Research. H.J.K. was a recipient of a Human Frontiers Postdoctoral fellowship and D.D. acknowledges support from EMBO through the Young Investigator Program (YIP).

Author information

Author notes

  1. Norimichi Nomura, Grégory Verdon and Hae Joo Kang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, 606-8501, Kyoto, Japan

    Norimichi Nomura, Tatsuro Shimamura, Yayoi Nomura, Yumi Sato, Hitomi Abe, Yoshiko Nakada-Nakura, Tomoya Hino, Takatoshi Arakawa, Takeshi Murata, Takuya Kobayashi & So Iwata

  2. Japan Science and Technology Agency, ERATO, Iwata Human Receptor Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, 606-8501, Kyoto, Japan

    Norimichi Nomura, Tatsuro Shimamura, Yayoi Nomura, Tomoya Hino, Takatoshi Arakawa, Takeshi Murata, Takuya Kobayashi & So Iwata

  3. Japan Science and Technology Agency, Research Acceleration Program, Membrane Protein Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, 606-8501, Kyoto, Japan

    Norimichi Nomura, Tatsuro Shimamura, Yayoi Nomura, Yumi Sato, Yoshiko Nakada-Nakura, Takeshi Murata, Takuya Kobayashi & So Iwata

  4. Division of Molecular Biosciences, Imperial College London, London, SW7 2AZ, UK

    Grégory Verdon, Hae Joo Kang, Yo Sonoda, So Iwata & David Drew

  5. Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Chilton, OX11 0DE, Oxfordshire, UK

    Grégory Verdon, Hae Joo Kang & So Iwata

  6. Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Didcot, OX11 0FA, Oxford, Oxfordshire, UK

    Grégory Verdon, Hae Joo Kang & So Iwata

  7. Department of Biochemistry and Biophysics, Centre for Biomembrane Research, Stockholm University, Stockholm, SE-106 91, Sweden

    Saba Abdul Hussien, Aziz Abdul Qureshi, Mathieu Coincon & David Drew

  8. Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, 153-8904, Tokyo, Japan

    Osamu Kusano-Arai, Hiroko Iwanari & Takao Hamakubo

  9. Systems and Structural Biology Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, 230-0045, Yokohama, Japan

    Takeshi Murata & So Iwata

  10. Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, 192-0395, Tokyo, Japan

    Michihiro Kasahara

Authors
  1. Norimichi Nomura
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  2. Grégory Verdon
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  3. Hae Joo Kang
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Contributions

N.N., S.I. and D.D. designed the project. Cloning, expression screening and initial crystallization of rat and bovine GLUT5 was carried out by H.J.K., Y.So. and D.D. Crystal optimization of bovine GLUT5 was carried out by H.J.K. and G.V. Data collection, structure determination and refinement of bovine GLUT5 was carried out by G.V. Generation of rat GLUT5 scFv fragment was carried out by N.N., Y.N., T.M., Y.N.-N., O.K.-A., H.I., T.A., T.K. and T.Ha. Expression and purification of the Fv fragment was carried out by N.N., Y.N., Y.Sa., H.A. and T.Hi. Co-crystallization of rat GLUT5–Fv complex and data collection was performed by N.N. and Y.N. with assistance from T.Hi. and S.I. Structure determination and refinement of rat GLUT5–Fv was carried out by T.S. Experiments for functional analysis were designed by M.K. and D.D. and carried out by M.K., D.D., S.A.H. and A.A.Q. Modelling of GLUT5 was carried out by M.C. The manuscript was prepared by N.N., H.J.K., G.V., S.I. and D.D. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Norimichi Nomura, So Iwata or David Drew.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Anisotropy descriptors of bGLUT5 data reported by the UCLA-MBI Diffraction Anisotropy Server and 2Fo − Fc electron density maps for the bovine and rat GLUT5 structures.

a, Degree of anisotropy of bGLUT5 data, resolution limits for the 3 principal axes (left), and panel illustrating steps along correction of bGLUT5 data for anisotropy (right). b, Representative portions of the electron density map (1.5σ) for bGLUT5 overall model (left) and a close-up of the substrate binding site (right); residues highlighted are numbered based on rGLUT5 for the sake of clarity. c, Electron density (1.0σ) for rGLUT5 showing one of the inter-bundle salt-bridge clusters that form in the open outward-facing conformation.

Extended Data Figure 2 Superimposition of open inward-facing bGLUT5 and hGLUT1 structures, and comparison of the substrate-binding site in bGLUT5 and inward-facing XylE

a, Ribbon representation of inward-facing bGLUT5 (coloured as in Fig. 1a) and inward-facing hGLUT1 (light grey) structures, as viewed in the plane of the membrane. The d-glucopyranoside moiety of the detergent molecule bound to GLUT1 (n-nonyl-β-d-glucopyranoside (β-NG)) is shown in stick representation. Density for ICH5 at the C terminus is missing in both hGLUT1 and bGLUT5 inward-facing structures and highlighted with the dotted ellipse. The beginning of TM1 kinks further outwards in the bGLUT5 structure compared to hGLUT1 and residues 1–18 could not be built. The r.m.s.d. (root mean square deviation) after superposition of the two structures is 1.12 Å for 364 pairs of Cα atoms (see Methods). b, The substrate-binding in the inward-facing bGLUT5 structure (coloured as in Fig. 1) is very similar to that seen in inward-facing XylE (4JA4) structure (shown in light grey). Only non-conserved residues and the equivalent glutamine to Q166 are labelled for XylE.

Extended Data Figure 3 Structure of the rat GLUT5–Fv complex.

a, Cartoon representation of the complex between rGLUT5 (grey) and 4D111Fv (heavy-chain variable region (VH) is in blue; light-chain variable region (VL) is in red). 4D111Fv binds to the cytoplasmic domain of GLUT5, including ICH2 (residues 226, 230, 234), the loop between ICH2 and ICH3 (residues 238, 240, 241), and ICH3 (residue 243), with ∼ 848 Å2 of buried surface area at the interface. b, Packing of the rat GLUT5–Fv complex molecules in the crystal. The unit cell is represented as green lines.

Extended Data Figure 4 Sequence alignment of rat GLUT5 (rGLUT5), bovine GLUT5 (bGLUT5), human GLUT5 and GLUT7 (hGLUT5 and hGLUT7), human GLUT1–4 (hGLUT1–4), Saccharomyces cerevisiae HXT7, Plasmodium falciparum PfHT), Arabidopsis thaliana GlcT and Escherichia coli XylE.

Structure elements of rat GLUT5 are indicated above the alignment, and coloured as in Fig. 1a. Strictly conserved residues are highlighted in black-filled boxes, and highly conserved residues are shaded in grey. Green boxes highlight central cavity residues that are specific to GLUT5 and red boxes highlight those that are conserved among GLUTs. Purple boxes highlight residues forming the salt bridges between cytosolic TM segments. A blue box (TM5) highlights Gln166, whose mutation to glutamic acid, as present in GLUT7, weakens d-fructose binding but supports strong d-glucose binding in rGLUT5. The brown box (TM8) highlights Glu336 that is conserved across all the GLUTs and replaced with glutamic acid in XylE. Red bars underneath the alignment indicate the sugar porter (SP) family motifs18,19. Note that because bGLUT5 and hGLUT5 have an additional amino acid at position 8, their numbering differs from rGLUT5 by 1 amino acid. For clarity, bGLUT5 residues are labelled using rGLUT5 numbering.

Extended Data Figure 5 d-fructose binding monitored by tryptophan fluorescence quenching.

a, Cartoon representation of the outward-facing rGLUT5 structure, as viewed from the plane of the membrane with the colouring as shown in Fig. 1a. Atoms in all tryptophan residues are shown as spheres and tryptophan W419, whose fluorescence is quenched by substrate, is labelled. b, Emission fluorescence spectra for purified deglycosylated rGLUT5 wild-type-like mutant N50Y (referred to as WT), shown in the range of 320–360 nm with an excitation wavelength of 295 nm after the addition of 40 mM d-fructose (top), and 40 mM l-fructose (bottom). Emission fluorescence spectra for purified wild-type protein that had been previously incubated with the inhibitor HgCl2 is also shown for d-fructose (middle). c, Tryptophan fluorescence quenching (excitation 295 nm; emission 338 nm) after incubation of purified rGLUT5 N50Y with either 40 mM d-fructose (filled bar) or l-fructose, d-glucose, d-mannose, d-xylose or d-galactose as labelled (open bars). Tryptophan fluorescence quenching for purified wild-type protein that had been previously incubated with the inhibitor HgCl2 is also shown for d-fructose (open bar). d, As in c, but for rGLUT5 with a single tryptophan residue (W419), which contains the following mutations: N50Y, W70F, W191F, W239F, W265F, W275F, W338F and W370F. No tryptophan quenching was observed for d-fructose (5 mM HgCl2), l-fructose, d-glucose or d-galactose. In all experiments errors bars indicate s.e.m.; n = 3.

Extended Data Figure 6 Substrate specificity in GLUT5.

a, Time-dependent uptake of d-[14C]-fructose by rGLUT5 wild type (open squares and triangles) and the deglycosylated mutant N50Y (filled squares and triangles) in proteoliposomes incubated with or without the inhibitor HgCl2 as labelled. Non-specific uptake was estimated with 0.1 mM l-[14C]-glucose for wild type (filled circles) and the N50Y mutant (open circles). In all experiments errors bars represent a spread of duplicates. Inset shows SDS–PAGE analysis of the purified rat GLUT5 wild type and the deglycosylated N50Y mutant. b, Tryptophan fluorescence quenching (excitation 295 nm; emission 338 nm), after incubation of purified rat GLUT5 mutant (N50Y, W70F, W191F, W239F, W265F, W275F, W338F, W370F) that contains one single tryptophan residue, W419, with increasing concentrations of d-fructose (filled squares) and to the protein previously incubated with the inhibitor mercury chloride (open circles). c, Slab through the surface of the outward-facing rGLUT5 structure as viewed in the plane of membrane. The structure of substrate-bound XylE structure was further superimposed onto rGLUT5 and is shown here as a grey ribbon. In XylE, Trp392 (Trp388 in hGLUT1) is located at the bottom of the cavity (spheres; magenta) and coordinates d-xylose (stick form; yellow). In GLUT5, the equivalent residue is an alanine, making the cavity deeper. d, d-fructose binding as measured by tryptophan fluorescence quenching (excitation 295 nm; emission 338 nm) after incubation with 40 mM d-fructose for wild type (open bar), and TM7 mutations of Ile295 (interacts with TM10 residues) and Tyr296 and Tyr297 residues. Equivalently located tyrosine residues in XylE occlude the sugar-binding site from the outside22. Fluorescence quenching for the mutants are displayed as a percentage of total wild-type binding. In all experiments errors bars indicate s.e.m.; n = 3.

Extended Data Figure 7 The intracellular helical domain (ICH).

a, Cytoplasmic view of the ICH domain after superposition of the open, outward-facing rGLUT5–sFv (grey) and outward-facing occluded E. coli XylE (teal) (4GBY) structures. b, In the outward-facing GLUT5 structure ICH1–ICH3 are linked together by several salt bridges (side chains are labelled and shown as sticks in yellow). In contrast, no polar interactions are formed between ICH5 and either ICH1–ICH3 or cytoplasmic ends of N-terminal TM bundle helices. A salt bridge forms (dotted line in magenta), however, between Glu225 in ICH3 and Arg407 in TM11, which also forms part of the inter-bundle salt-bridge network (side chains are labelled and shown as sticks in cyan). c, In the inward-facing GLUT5 structure, this inter-bundle salt-bridge network is not formed, because the cytoplasmic ends of the N- and C-terminal bundle have moved apart; consistently, the ICH domain functional role is proposed to act as a scaffold domain that further helps to stabilize the outward-facing conformation21.

Extended Data Figure 8 Access to the central cavity and substrate-binding site is gated by TM7 on the outside and TM10 on the inside.

a, Superposition of outward-facing open GLUT5 and outward-facing occluded E. coli XylE (4GBY) structures. The TM numbering for outward-facing occluded XylE has an additional asterisk. The inward-facing GLUT5 structure is coloured as in Fig. 1a and that of XylE in grey. The bound d-xylose is shown in stick representation in green. The r.m.s.d. is 1.38 Å for 290 pairs of Cα atoms (see Methods). b, Superposition of inward-open GLUT5 and inward-occluded E. coli XylE structure (4JA3) with colouring and annotation as described in a. The r.m.s.d. is 1.80 Å for 274 pairs of Cα atoms (see Methods). The bound d-xylose in 4GBY is represented in stick form in green. The ICH domain is not shown for clarity. c, Superposition of inward-facing open GLUT5 and inward-facing open XylE (4JA4) structures as viewed from the cytoplasmic side with colouring and annotation as described in a. The ICH domain is not shown for clarity. The r.m.s.d. is 1.70 Å for 273 pairs of Cα atoms (see Methods).

Extended Data Table 1 Crystallographic data collection and refinement statistics
Full size table
Extended Data Table 2 Completeness of bovine GLUT5 data per resolution shell after correction for anisotropy
Full size table

Supplementary information

Video 1: Alternating access model of GLUT5 transport

This video shows morphing between the open outward-facing, outward occluded, inward-occluded and open inward-facing GLUT5; conformations as viewed parallel to the membrane. The open outward- and inward-facing conformations are based on the GLUT5 structures reported in this study, and occluded states were modeled based on homologous XylE crystal structures (Methods). Morphing between conformations was generated using PyMol. Helix colouring is as in Fig. 1a. (MOV 20436 kb)

Video 2: Alternating access model of GLUT5 transport

This video shows the same as Video 1, but viewed from the extracellular side of the membrane. (MOV 17095 kb)

Video 3: Alternating access model of GLUT5 transport

This video shows the same as Video 1, but viewed from the intracellular side of the membrane. (MOV 16468 kb)

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Nomura, N., Verdon, G., Kang, H. et al. Structure and mechanism of the mammalian fructose transporter GLUT5. Nature 526, 397–401 (2015). https://doi.org/10.1038/nature14909

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