Crystal structure of the human glucose transporter GLUT1

Journal name:
Nature
Volume:
510,
Pages:
121–125
Date published:
DOI:
doi:10.1038/nature13306
Received
Accepted
Published online

Abstract

The glucose transporter GLUT1 catalyses facilitative diffusion of glucose into erythrocytes and is responsible for glucose supply to the brain and other organs. Dysfunctional mutations may lead to GLUT1 deficiency syndrome, whereas overexpression of GLUT1 is a prognostic indicator for cancer. Despite decades of investigation, the structure of GLUT1 remains unknown. Here we report the crystal structure of human GLUT1 at 3.2Å resolution. The full-length protein, which has a canonical major facilitator superfamily fold, is captured in an inward-open conformation. This structure allows accurate mapping and potential mechanistic interpretation of disease-associated mutations in GLUT1. Structure-based analysis of these mutations provides an insight into the alternating access mechanism of GLUT1 and other members of the sugar porter subfamily. Structural comparison of the uniporter GLUT1 with its bacterial homologue XylE, a proton-coupled xylose symporter, allows examination of the transport mechanisms of both passive facilitators and active transporters.

At a glance

Figures

  1. Overall structure of the human glucose transporter GLUT1.
    Figure 1: Overall structure of the human glucose transporter GLUT1.

    The structure of full-length human GLUT1 containing two point mutations (N45T, E329Q) was determined in an inward-open conformation. The side and cytoplasmic views are shown. The corresponding transmembrane segments in the four 3-helix repeats are coloured the same. The extracellular and intracellular helices are coloured blue and orange, respectively. A slab of cut-open view of the surface electrostatic potential, which was calculated with PyMol50, is shown on the right to facilitate visualization of the inward-facing cavity. IC indicates intracellular helix. All structure figures were prepared with PyMol.

  2. Structural mapping of disease-derived mutations in GLUT1.
    Figure 2: Structural mapping of disease-derived mutations in GLUT1.

    The residues for which mutations have been identified in patients with GLUT1 deficiency syndrome and other symptoms are coloured magenta. The orange circle and the grey and cyan shades indicate the three major clusters of residues for which mutations are associated with diseases. The N, C and ICH domains are coloured green, blue and yellow, respectively. The same colour scheme is used in all subsequent figures. Details of the mutations can be found in Extended Data Table 2.

  3. The ICH domain serves as a latch that tightens the intracellular gate.
    Figure 3: The ICH domain serves as a latch that tightens the intracellular gate.

    a, Structural comparison of the inward-open GLUT1 to its E. coli homologue XylE (PDB accession code 4GC0)28. b, Structural overlay of GLUT1 and XylE using their respective N domains. For visual clarity, GLUT1 is displayed in three colours and XylE is coloured grey. c, Analysis of the disease-related GLUT1 residues that mediate the inter-domain interactions on the intracellular side. Left: positioning and interactions of the disease-related residues on the intracellular side of the inward-open GLUT1. Right: the interaction network of the corresponding residues in the outward-facing XylE. The residues shown in this panel are conserved between GLUT1 and XylE, with the disease-related residues coloured and labelled magenta. The XylE residues are numbered according to their counterparts in GLUT1. Hydrogen bonds are represented by dashed lines.

  4. Analysis of the extracellular gate of the inward-open GLUT1.
    Figure 4: Analysis of the extracellular gate of the inward-open GLUT1.

    a, TM1 and TM7 represent the major constituents of the extracellular gate in the inward-open GLUT1. b, Several disease-related residues mediate the interactions between the N and C domains on the extracellular side of the inward-open GLUT1. A close-up view of the hydrogen bond network is shown in the inset. c, Comparison of the local interactions for the disease-related residue Arg126 in GLUT1 and the corresponding residue Arg133 in XylE. The residues of XylE are labelled according to their counterparts in GLUT1 whereas their native numbers are indicated in brackets. d, Arg126 on TM4 of GLUT1 may form a cation–π interaction with Tyr292 on TM7 of the C domain, contributing to the affinity of the extracellular gate. The structures of GLUT1 and XylE are superimposed using their respective N domains.

  5. A working model for GLUT1.
    Figure 5: A working model for GLUT1.

    Shown here are the predicted conformations—outward-open, ligand-bound and occluded, inward-open, and ligand-free and occluded—required for a complete transport cycle according to the alternating access model. The inward-open conformation of GLUT1 is reported in this study. The ligand-bound, occluded conformation and the ligand-free, occluded one are predicted from two XylE structures in the outward-facing, partly occluded and ligand-bound state28, and the inward-facing, occluded state29, respectively. The outward-open structure remains to be captured. The ICH domain is illustrated as a latch that strengthens the intracellular gate in the outward-facing conformations. The extracellular gate comprises a few residues from TM1, TM4 and TM7 that are illustrated by the red brick in the ‘inward open’ cartoon.

  6. Structure determination of GLUT1.
    Extended Data Fig. 1: Structure determination of GLUT1.

    a, The 2FoFc electron density map. The stereo-view map for one representative slab, shown as cyan mesh, is contoured at 1.0σ. b, The crystal packing of GLUT1 in the space group C2. Each GLUT1 molecule is shown as rainbow-coloured ribbon, blue and red for the N and C termini, respectively.

  7. One [bgr]-NG molecule occupies the substrate-binding site of the inward-open GLUT1.
    Extended Data Fig. 2: One β-NG molecule occupies the substrate-binding site of the inward-open GLUT1.

    a, The ‘omit’ electron density observed in the inward-open cavity of GLUT1. The electron density, shown as magenta mesh, is contoured at 3.0σ. The N, C and ICH domains are coloured green, blue and yellow, respectively. b, A β-NG molecule fits well into the electron density inside the cavity. The 2FoFc electron density map (cyan mesh) for the β-NG molecule is contoured at 1.0σ. c, The overall GLUT1 structure with the bound β-NG molecule. β-NG is represented by white spheres. d, The coordination of the sugar moiety of β-NG by GLUT1 is similar to the binding of d-glucose by XylE. The structures of GLUT1 (blue) and XylE (cyan) are superimposed relative to their respective C domains. The ligands are shown in stick representation. Despite the similarity between d-glucose and β-NG, we cannot exclude the possibility that presence of the aliphatic tail of β-NG in GLUT1 may subtly affect positioning of the sugar moiety compared to d-glucose. e, Coordination of the d-glucopyranoside of β-NG by GLUT1. The d-glucopyranoside of β-NG is hydrogen-bonded to the surrounding polar residues in the C domain, including Gln282/Gln283/Asn288 from TM7, Asn317 from TM8, and Asn415 from TM11. The residues whose mutations are associated with GLUT1 deficiency syndrome are labelled in magenta.

  8. Interactions between the N and C domains observed in the inward-open GLUT1 and the outward-facing XylE.
    Extended Data Fig. 3: Interactions between the N and C domains observed in the inward-open GLUT1 and the outward-facing XylE.

    a, In GLUT1, the inter-domain contacts, mainly on the extracellular side, include both van der Waals interactions and hydrogen bonds. Residues whose mutations are associated with GLUT1 deficiency syndrome are labelled in magenta. b, In XylE, there are only limited interactions between the N and C domains at the transmembrane region.

  9. Conformational differences between GLUT1 and XylE.
    Extended Data Fig. 4: Conformational differences between GLUT1 and XylE.

    a, Structural comparison of the inward-open GLUT1 and the outward-facing, partly occluded, ligand-bound XylE. Similar intracellular views are shown for GLUT1 and XylE. Note that the C-terminal helix IC5, which was referred to as IC4 in a previous study of XylE (ref. 28), is invisible in the structure of GLUT1 probably due to its inherent flexibility in this conformation. b, c, Structural superimpositions of GLUT1 and XylE relative to their respective N domains (b) and C domains (c). A detailed analysis can be found in Figs 3 and 4. d, Conformational differences of TM7 between GLUT1 and XylE. Compared to that in XylE, the extracellular segment of TM7 in GLUT1 is further bent away from the transport path.

  10. Sequence alignment of GLUT1-4 with XylE.
    Extended Data Fig. 5: Sequence alignment of GLUT1-4 with XylE.

    Secondary structural elements of GLUT1 are indicated above the sequence alignment. Invariant and highly conserved amino acids are shaded yellow and grey, respectively. The conserved sugar porter family signature motifs are underscored with red lines. The residues that are hydrogen-bonded to d-glucose in XylE are shaded red. The GLUT1 residues whose mutations were found in GLUT1 deficiency syndrome are indicated by black circles above. The sequences were aligned with ClustalW.

Tables

  1. Statistics of data collection and refinement for GLUT1
    Extended Data Table 1: Statistics of data collection and refinement for GLUT1
  2. Summary of disease-related sequence variations of GLUT1
    Extended Data Table 2: Summary of disease-related sequence variations of GLUT1

Accession codes

Primary accessions

Protein Data Bank

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Author information

  1. These authors contributed equally to this work.

    • Dong Deng,
    • Chao Xu,
    • Pengcheng Sun &
    • Jianping Wu

Affiliations

  1. State Key Laboratory of Bio-membrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China

    • Dong Deng,
    • Chao Xu,
    • Pengcheng Sun,
    • Jianping Wu,
    • Chuangye Yan,
    • Mingxu Hu &
    • Nieng Yan
  2. Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China

    • Dong Deng,
    • Chao Xu,
    • Pengcheng Sun,
    • Jianping Wu,
    • Chuangye Yan,
    • Mingxu Hu &
    • Nieng Yan
  3. Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China

    • Dong Deng,
    • Chao Xu,
    • Jianping Wu,
    • Mingxu Hu &
    • Nieng Yan

Contributions

N.Y. conceived the project. D.D. and N.Y. designed all experiments. D.D., C.X., P.S., J.W., C.Y. and M.H. performed the experiments. All authors analysed the data and contributed to manuscript preparation. N.Y. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The X-ray crystallographic coordinates and structure factor files of human GLUT1(N45T/E329Q) have been deposited in the Protein Data Bank (PDB) with the accession code 4PYP.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Structure determination of GLUT1. (964 KB)

    a, The 2FoFc electron density map. The stereo-view map for one representative slab, shown as cyan mesh, is contoured at 1.0σ. b, The crystal packing of GLUT1 in the space group C2. Each GLUT1 molecule is shown as rainbow-coloured ribbon, blue and red for the N and C termini, respectively.

  2. Extended Data Figure 2: One β-NG molecule occupies the substrate-binding site of the inward-open GLUT1. (829 KB)

    a, The ‘omit’ electron density observed in the inward-open cavity of GLUT1. The electron density, shown as magenta mesh, is contoured at 3.0σ. The N, C and ICH domains are coloured green, blue and yellow, respectively. b, A β-NG molecule fits well into the electron density inside the cavity. The 2FoFc electron density map (cyan mesh) for the β-NG molecule is contoured at 1.0σ. c, The overall GLUT1 structure with the bound β-NG molecule. β-NG is represented by white spheres. d, The coordination of the sugar moiety of β-NG by GLUT1 is similar to the binding of d-glucose by XylE. The structures of GLUT1 (blue) and XylE (cyan) are superimposed relative to their respective C domains. The ligands are shown in stick representation. Despite the similarity between d-glucose and β-NG, we cannot exclude the possibility that presence of the aliphatic tail of β-NG in GLUT1 may subtly affect positioning of the sugar moiety compared to d-glucose. e, Coordination of the d-glucopyranoside of β-NG by GLUT1. The d-glucopyranoside of β-NG is hydrogen-bonded to the surrounding polar residues in the C domain, including Gln282/Gln283/Asn288 from TM7, Asn317 from TM8, and Asn415 from TM11. The residues whose mutations are associated with GLUT1 deficiency syndrome are labelled in magenta.

  3. Extended Data Figure 3: Interactions between the N and C domains observed in the inward-open GLUT1 and the outward-facing XylE. (577 KB)

    a, In GLUT1, the inter-domain contacts, mainly on the extracellular side, include both van der Waals interactions and hydrogen bonds. Residues whose mutations are associated with GLUT1 deficiency syndrome are labelled in magenta. b, In XylE, there are only limited interactions between the N and C domains at the transmembrane region.

  4. Extended Data Figure 4: Conformational differences between GLUT1 and XylE. (460 KB)

    a, Structural comparison of the inward-open GLUT1 and the outward-facing, partly occluded, ligand-bound XylE. Similar intracellular views are shown for GLUT1 and XylE. Note that the C-terminal helix IC5, which was referred to as IC4 in a previous study of XylE (ref. 28), is invisible in the structure of GLUT1 probably due to its inherent flexibility in this conformation. b, c, Structural superimpositions of GLUT1 and XylE relative to their respective N domains (b) and C domains (c). A detailed analysis can be found in Figs 3 and 4. d, Conformational differences of TM7 between GLUT1 and XylE. Compared to that in XylE, the extracellular segment of TM7 in GLUT1 is further bent away from the transport path.

  5. Extended Data Figure 5: Sequence alignment of GLUT1-4 with XylE. (746 KB)

    Secondary structural elements of GLUT1 are indicated above the sequence alignment. Invariant and highly conserved amino acids are shaded yellow and grey, respectively. The conserved sugar porter family signature motifs are underscored with red lines. The residues that are hydrogen-bonded to d-glucose in XylE are shaded red. The GLUT1 residues whose mutations were found in GLUT1 deficiency syndrome are indicated by black circles above. The sequences were aligned with ClustalW.

Extended Data Tables

  1. Extended Data Table 1: Statistics of data collection and refinement for GLUT1 (275 KB)
  2. Extended Data Table 2: Summary of disease-related sequence variations of GLUT1 (225 KB)

Additional data