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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.
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 (2.6â3.6 Ã distances). The omit Fo-Fc density for NG is contoured in green at 3 Ï. (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 Ï (top). The anomalous signal for the chemical chloride congener, bromide, shown in magenta mesh, is contoured at 4 Ï (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.
Adams,
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
2010, Pubmed
Adams,
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
2010,
Pubmed
Bailey,
MEME: discovering and analyzing DNA and protein sequence motifs.
2006,
Pubmed
Baldwin,
Mammalian passive glucose transporters: members of an ubiquitous family of active and passive transport proteins.
1993,
Pubmed
Bentley,
Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes.
2012,
Pubmed
,
Xenbase
Berend,
Chloride: the queen of electrolytes?
2012,
Pubmed
Bond,
ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments.
2009,
Pubmed
Burant,
Mammalian facilitative glucose transporters: evidence for similar substrate recognition sites in functionally monomeric proteins.
1992,
Pubmed
,
Xenbase
Cain,
The conserved motif in hydrophilic loop 2/3 and loop 8/9 of the lactose permease of Escherichia coli. Analysis of suppressor mutations.
2000,
Pubmed
Chen,
MolProbity: all-atom structure validation for macromolecular crystallography.
2010,
Pubmed
Deng,
Molecular basis of ligand recognition and transport by glucose transporters.
2015,
Pubmed
Deng,
Crystal structure of the human glucose transporter GLUT1.
2014,
Pubmed
De Zutter,
Sequence determinants of GLUT1 oligomerization: analysis by homology-scanning mutagenesis.
2013,
Pubmed
Ekberg,
Structural identification of cation binding pockets in the plasma membrane proton pump.
2010,
Pubmed
Emsley,
Features and development of Coot.
2010,
Pubmed
Hresko,
Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids.
2016,
Pubmed
Iancu,
Crystal structure of a glucose/H+ symporter and its mechanism of action.
2013,
Pubmed
Jiang,
Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A.
2013,
Pubmed
Kabsch,
XDS.
2010,
Pubmed
Kapoor,
Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.
2016,
Pubmed
Kidmose,
Namdinator - automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps.
2019,
Pubmed
Leduc-Nadeau,
Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes.
2007,
Pubmed
,
Xenbase
Lowe,
The kinetics of glucose transport in human red blood cells.
1986,
Pubmed
Lyons,
Expression strategies for structural studies of eukaryotic membrane proteins.
2016,
Pubmed
Maher,
Substrate specificity and kinetic parameters of GLUT3 in rat cerebellar granule neurons.
1996,
Pubmed
Martens,
Direct protein-lipid interactions shape the conformational landscape of secondary transporters.
2018,
Pubmed
McCoy,
Phaser crystallographic software.
2007,
Pubmed
Mueckler,
Sequence and structure of a human glucose transporter.
1985,
Pubmed
Mumberg,
Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression.
1994,
Pubmed
Nishimura,
Kinetics of GLUT1 and GLUT4 glucose transporters expressed in Xenopus oocytes.
1993,
Pubmed
,
Xenbase
Nomura,
Structure and mechanism of the mammalian fructose transporter GLUT5.
2015,
Pubmed
Paulsen,
Crystal structure of the plant symporter STP10 illuminates sugar uptake mechanism in monosaccharide transporter superfamily.
2019,
Pubmed
,
Xenbase
Pei,
PROMALS3D: a tool for multiple protein sequence and structure alignments.
2008,
Pubmed
Saier,
The transporter classification database.
2014,
Pubmed
Sato,
A conserved amino acid motif (R-X-G-R-R) in the Glut1 glucose transporter is an important determinant of membrane topology.
1999,
Pubmed
,
Xenbase
Seyfang,
Four conserved cytoplasmic sequence motifs are important for transport function of the Leishmania inositol/H(+) symporter.
2000,
Pubmed
,
Xenbase
Simpson,
The facilitative glucose transporter GLUT3: 20 years of distinction.
2008,
Pubmed
Sobolev,
A Global Ramachandran Score Identifies Protein Structures with Unlikely Stereochemistry.
2020,
Pubmed
Sun,
Crystal structure of a bacterial homologue of glucose transporters GLUT1-4.
2012,
Pubmed
Tomasiak,
General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays.
2014,
Pubmed
Wheeler,
Infinite-cis kinetics support the carrier model for erythrocyte glucose transport.
1988,
Pubmed
Wheeler,
Characterization of glucose transport activity reconstituted from heart and other tissues.
1998,
Pubmed
Winn,
Overview of the CCP4 suite and current developments.
2011,
Pubmed
Wisedchaisri,
Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE.
2014,
Pubmed
Zhang,
Uniporter substrate binding and transport: reformulating mechanistic questions.
2016,
Pubmed
Zhang,
Energy coupling mechanisms of MFS transporters.
2015,
Pubmed
