Custódio TF et al. (2021), Structural comparison of GLUT1 to GLUT3 reveal ...

XB-ART-58520

Life Sci Alliance 2021 Feb 03;44:. doi: 10.26508/lsa.202000858.

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Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in sugar porter family.

Custódio TF , Paulsen PA , Frain KM , Pedersen BP .

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The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic "SP motif" and a conserved "A motif" stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl- ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.

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Species referenced: Xenopus laevis
Genes referenced: slc2a1 slc2a3
GO keywords: glucose binding [+]

???displayArticle.disOnts??? dystonia 9 [+]
???displayArticle.omims??? DYSTONIA 9; DYT9 [+]

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	Figure 1. Comparison between GLUT1 and GLUT3. (A) Schematic model for transport by GLUTs, alternating between two major conformations with the substrate-binding site exposed to the inside and outside of the cell. Transition between these conformations leads to sugar transport across the membrane following the substrate concentration gradient. (B) Uptake of 2-DG into GLUT1-injected Xenopus oocytes (circle), GLUT3-injected oocytes (squares) or water-injected oocytes (open triangles) at an initial outside concentration of 5 mM 2-DG. For both proteins, 2-DG uptake was linear in the range of 90 min. Data for all assays are mean ± SD of three or more replicate experiments. (C) Determination of the kinetic parameters for the transport of 2-DG of GLUT1. The data were fitted using the Michaelis–Menten non-linear fit, yielding a Km = 9.5 ± 1.0 mM and Vmax = 5,988 ± 226 pmol/oocyte/30 min. Sugar uptake was inhibited in GLUT1-injected oocytes exposed to cytochalasin B. (D) Determination of the kinetic parameters for the transport of 2-DG of GLUT3. The data were fitted using the Michaelis–Menten non-linear fit, yielding a Km = 2.6 ± 0.4 mM and Vmax = 2,731 ± 94 pmol/oocyte/30 min. Sugar uptake was inhibited in GLUT3-injected oocytes exposed to cytochalasin B. (E) Substrate selectivity of GLUT1 determined by competition assay in oocytes exposed to 5 mM 2-DG and 20× fold of the competing sugar, for 15 min. (F) Substrate selectivity of GLUT3 determined by competition assay in oocytes exposed to 5 mM 2-DG and 20× fold of the competing sugar, for 15 min. Data information: In (B, C, D, E, F) Data for all assays are mean ± SD of three or more replicate experiments. In (E, F) ns, Not significant; P ≤ 0.05; P ≤ 0.01; and *P ≤ 0.001 by t test. P-value is shown for ns and .
	Figure 2. Crystal structure of GLUT1 reveals new ligands. (A) The overall structure of GLUT1 in the inward-open conformation. The structure represents a bound state with an NG molecule (shown as sticks) in the central cavity formed between the N-domain (blue) and the C-domain (brown), followed by a PEG molecule (shown as sticks). In close proximity with the ICH domain (yellow) another NG molecule (shown as sticks) was found, as well as a chloride ion (shown as a sphere). Selected residues are shown as sticks. Black lines depict the approximate location of the membrane. (B) Coordination of the glucose moiety in the central cavity by residues from C-domain. Hydrogen bonds are represented by yellow dashes (2.6–3.6 Å distances). The omit Fo-Fc density for NG and PEG is contoured in green at 3 σ. (C) Side view of GLUT1 shows the localization of the N-domain Sugar Porter motif directly underneath the A-motif from the M2-M3 loop. Signature motifs are shown as spheres. (D) Side view of GLUT1 shows the localization of the C-domain Sugar Porter motif directly underneath the A-motif from the M8-M9 loop. Signature motifs are shown as spheres.
	Figure 3. Intracellular binding of Chloride and NG stabilize the inward-open conformation. (A) Intracellular NG is coordinated by residues from the Sugar Porter and A motifs. A chloride ion (shown as spheres) neutralizes the A-motif. Hydrogen bonds are represented by yellow dashes (2.6–3.6 Å distances). The omit Fo-Fc density for NG is contoured in green at 3 σ. (B) Coordination of the chloride ion by residues of the A-motif. The Fo-Fc electron density, shown in green mesh, is contoured at 4 σ (top). The anomalous signal for the chemical chloride congener, bromide, shown in magenta mesh, is contoured at 4 σ (bottom).
	Figure 4. The N-domain Sugar Porter (SP)-A network between inward and outward states. (A) Sequence alignment between GLUT1 and GLUT3 of the N-domain A motif (blue) and the N-domain SP motif (yellow). Residues involved in GLUT1 deficiency syndrome are colored in red. N-domain SP-A network in the inward conformation represented by the GLUT1 structure (left) and the outward conformation represented by the GLUT3 structure (PDB 4ZW9) (right). Selected residues are shown as sticks and hydrogen bonds are represented by yellow dashes (2.6–3.6 Å distances). (B) GLUT1 E209Q: Km = 17 ± 3.4 mM and Vmax = 6,473 ± 528 pmol/oocyte/30 min. (C) GLUT3 E207Q: Km = 6.2 ± 1.2 mM and Vmax = 1,418 ± 88 pmol/oocyte/30 min. Data information: In (B, C) Michaelis–Menten analysis of 2-DG uptake in oocytes. Data represents the mean ± SD of three or more replicate experiments.
	Figure 5. The C-domain Sugar Porter (SP)-A network between inward and outward states. (A) Sequence alignment between GLUT1 and GLUT3 of the C-domain A motif (brown) and the C-domain SP motif (yellow). Residues involved in GLUT1 deficiency syndrome are colored in red. C-domain SP-A network in the inward conformation represented by the GLUT1 structure (left) and the outward conformation represented by the GLUT3 structure (PDB 4ZW9) (right). Selected residues are shown as sticks and hydrogen bonds are represented by yellow dashes (2.6–3.6 Å distances). (B) GLUT1 K456R: Km = 4.2 ± 0.6 mM and Vmax = 2,427 ± 90 pmol/oocyte/30 min. (C) GLUT3 R454K: Km = 17 ± 3.3 mM and Vmax = 2,638 ± 234 pmol/oocyte/30 min. Data information: In (B, C) Michaelis–Menten analysis of 2-DG uptake in oocytes. Data represent the mean ± SD of three or more replicate experiments.
	Figure 6. Model for kinetic control of the transport cycle based on the Sugar Porter (SP)-A network. The SP-A network stabilizes the outward conformation where glucose binds from the extracellular side to the central substrate binding site. Glucose binding leads to closure of the binding site towards the extracellular side and the SP motif glutamate flips away from the A-motif, interchanged by a chloride ion. The disruption of the SP-A network opens the central cavity to the intracellular side and a cytosolic exit pathway for glucose is created. Direct interactions between glucose/lipids with the SP and A motifs can stabilize the inward conformation.

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