Glucose transporters are essential for metabolism of glucose in cells of diverse organisms from microbes to humans. Most of the important amino acids responsible for recognition of D-xylose or D-glucose are invariant in GLUT1–4, suggesting functional and mechanistic conservations. 7
Glucose provides energy in the form of ATP through glycolysis and the citric acid cycle and reducing power in the form of NADPH though the pentose phosphate shunt. It is also used in the synthesis of glycerol for triglyceride production and provides intermediates for synthesis of non-essential amino acids. 11
Complex carbohydrates represent one of the four major macromolecular structural building blocks of all living organisms. They also serve as primary storage sources of carbon and energy synthesized by and available to almost all living cells. Glucose is the most stable and most prevalent monosaccharide found in nature, and both cellulose and glycogen, structural and storage carbohydrates, respectively, consisting exclusively of glucose, comprise significant fractions of the biomass on earth. 5 The first step in the catabolism of an exogenous carbon source is usually transport of the molecule across a cell membrane.
Even simple bacteria may depend on more than one transport system to allow access of cytoplasmic enzymes to an exogenous carbon source. Thus, Gramnegative bacteria must transport a potential carbohydraterich energy source across both membranes of its cell envelope, and the molecular species transported across the outer membrane may differ from that that eventually enters the cytoplasm because of either hydrolytic activities present in the periplasmic space between these two membranes or the action of group translocating permeases that phosphorylate sugars during transport.
Escherichia coli can transport glucose-6-P from the medium across its outer membrane via porins, and the resultant periplasmic glucose-6-P can either be taken up into the cytoplasm directly via a hexose phosphate uptake permease, or be hydrolysed. If hydrolysed, the resultant free sugar and inorganic phosphate can be taken up into the cytoplasm via two distinct transport systems.
Interestingly, the mammalian microsomal transport protein is similar in sequence to that found in E. coli. Both proteins cluster tightly together on a phylogenetic tree, which groups proteins according to their degrees of sequence similarity. They are members of a family called the organophosphate: inorganic phosphate antiporter (OPA) family within one of the two largest superfamilies of transporters found in living organisms, a group of proteins called the major facilitator superfamily (MFS)
The common sugar glucose is a building block of carbohydrates. Glucose supplies energy to make ATP. 3 Respiration liberates energy by oxidizing glucose into carbon dioxide and water.
Glucose is a basic nutrient in most of the creatures; its transport through biological membranes is an absolute requirement of life. This role is fulfilled by glucose transporters, mediating the transport of glucose by facilitated diffusion or by secondary active transport.1 A first step before the catabolism of an exogenous sugar is usually transported across a cell membrane, and the exchange of sugars between different cells in organisms makes sugar transport a critical process. 2 Organisms have several mechanisms to transport sugars across membranes. These transporters can be classified into mainly three superfamilies:
major facilitator superfamily (MFS) transporters,
sodium-solute symporter family (SSF) transporters,
(SWEETs) and SemiSWEETs families
MFS transporters are conserved from bacteria to humans and include the
bacterial lac permease,
yeast hexose transporters (HXTs),
human glucose transporters (GLUTs),
plant sugar transporters.
SSF proteins catalyze sugar uptake across the cytoplasmic membranes of pro- and eukaryotic cells via the electrochemical sodium gradient (sodium motive force).
SWEETs catalyze the facilitated diffusion of sugars driven by their concentration gradients. The SWEETs in eukaryotes typically consist of seven transmembrane helices (TMHs), including a pair of 3-TMH repeats and an additional helix connecting these two repeats. In contrast, bacterial SemiSWEETs possess 3 TMHs in total.
The protein family of facilitative glucose transporters comprises 14 isoforms that share common structural features such as 12 transmembrane domains, N- and C-termini facing the cytoplasm of the cell, and a N-glycosylation side either within the first or fifth extracellular loop. Based on their sequence homology, three classes can be distinguished: class I includes GLUT1-4 and GLUT14, class II the ‘‘odd transporters’’ GLUT5, 7, 9, 11, and class III the ‘‘even transporters’’ GLUT6, 8, 10, 12 and the proton driven myoinositol transporter HMIT (or GLUT13). 6
New insights into GluT1 mechanics during glucose transfer 30 January 2019
All the MFS proteins share a common fold: 12 transmembrane (TM) helices are organized in two distinct domains called N- and C-domains, each consisting of six consecutive helices. Located against each other taking the form of a “V”, these domains form a large hydrophilic cavity at the center of the protein. The amino acids located inside the cavity ensure the ligand binding and thus determine the substrate specificity of the transporter5. Opening of the protein cavity to the cytoplasm or periplasm/extracellular medium allows the solute transfer by the MFS proteins. According to the model of the alternating access mechanism6, the cycle of the conformational changes of the MFS proteins during the ligand transport includes the outward facing (open to the extracellular medium) conformation necessary for the ligand uptake, the transitional closed states with the ligand located in the protein cavity isolated from the both extracellular and intracellular media, and the inward facing (open to cytoplasm) state allowing the ligand release.
Finally, the glucose transfer accompanied by the protein transition from the outward facing to the inward facing state is a highly cooperative process and requires simultaneous rearrangement of the local and global protein structure induced by a solute.9
Functional architecture of MFS d-glucose transporters 4 February 18, 2014
The crystallographic model of the Major Facilitator Superfamily (MFS) member, D-xylose permease (XylE) from Escherichia coli, a homologue of human D-glucose transporters GLUTs (SLC2), provides a structural framework for the identification and physical localization of crucial residues in transporters.
The Major Facilitator Superfamily (MFS) is a diverse group of secondary transporters with over 10,000 members, found in all kingdoms of life, including Homo sapiens.
XylE and GLUT1 contains a remarkably conserved sugar-binding site.
Glutamine 161 of Glutl Glucose Transporter Is Critical for Transport Activity and Exofacial Ligand Binding 10
The possible role of 5 transmembrane amino acid residues in the function of the Glutl glucose transporter was investigated by site-directed mutagenesis. The residues were chosen based on their containing hydroxyl or amide side chains capable of hydrogen bonding to glucose and their complete conservation in Glutl through Glut5. Asn1O0, Glnlel, Glnm , w", and were individually replaced by hydrophobic and/or polar residues, and the mutants were analyzed by expression in Xenopus oocytes. Substitution of leucine or asparagine for Gln"' reduced the relative transport activity of Glutl by 60- and 10-fold, respectively, as measured by uptake of 2-deoxyglucose normalized to the plasma membrane content of the mutant transporters.
Two observations indicate that GlnI6l is a particularly crucial amino acid residue for the function of the Glutl transporter. First, the activity of the transporter is strikingly reduced by mutations at this residue. A nonconservative substitution of leucine for the glutamine reduced transport activity by 50-fold, and a conservative substitution of asparagine for the glutamine reduced intrinsic activity by 10-fold. Second, the conservative substitution of asparagine for Gln161 reduced the apparent affinity of an exofacial ligand for the external binding site by 18-fold. Thus, the lack of a single side chain methylene group (the difference in structure between glutamine and asparagine) was sufficient to grossly perturb the function and ligand binding characteristics of Glutl. It thus seems unlikely that the severe disruption in transport activity observed for the Gln161 mutants was due to a major change in the tertiary structure of the molecule. Rather, these considerations suggest that the precise geometry of the glutamine side chain must be preserved for optimal transporter function. These data are consistent with GlP constituting an essential part of the exofacial substrate-binding site of the transporter
This is remarkable. The mutation/substitution of just ONE amino acids disrupts the glucose transport function. That demonstrates the high specificity required for the protein to bear function. Consider on top of that, that these import proteins are life essential, and had to emerge prior when life began!!
(A) Helices represented by colored boxes are shown in consecutive order in the sequence and colored according to helix-triplets. Arrows of the same color indicate the positions of correlating residues.
(B) Helix-triplets from XylE and LacY are aligned (helices 1–3, blue; helices 4–6, green; helices 7–9, orange; helices 10–12, yellow). The flags indicate the loops within triple-helix motifs. Helix-triplets from LacY and XylE are colored as in A. The alignments are oriented with the LacY cytoplasmic side at the top. The flags indicate loops within triple-helix motifs (white, cytoplasmic loop; gray, periplasmic loop). The numbers on the flags indicate the two helices that are connected by the respective loop.
(C) Schematic superposition. Helix-triplets are colored as in A. Overlapping side-chain positions are shown in the same color for corresponding helices. Contacts between side-chains are indicated as broken lines. Red boxes indicate positions lowering the transport activity of GLUT1 Cys-mutants; gray boxes indicate positions not tested by Cys-scanning mutagenesis in GLUT1. Yellow background indicates positions implicated in a medical condition.
Transport cycle of MFS symporter.
(A) Overview of the postulated steps in the transport model. Inward-facing (green) and outward-facing (blue) conformations are separated by the apo-intermediate conformations (orange) or by the occluded-intermediate conformations (gray). Steps are numbered consecutively: 1: Opening of the H+ site; 2: Deprotonation to inside and reorientation to the apo-intermediate with a central cavity closed to either side of the membrane; 3: Opening of the outward-facing cavity and reprotonation from the outside; 4: Formation of outward-open, substrate-free conformation; 5 and 6: Substrate binding and induced fit to the occluded conformation; 7: Opening of the inward-facing cavity and release of the sugar; 8: Formation of the protonated, substrate-free conformation.
(B) Hypothetical energy profile for the transport cycle. Conformational shown in A are translated into relative energy states (indicated by the icons of conformations defined in A with the cytoplasmic side of the symporter facing up). The schemes are cycles read by following the arrowheads. The red part of the cycle represents the transitions between steps 1 and 4 of the empty pathway. The blue line corresponds to steps 5–8 for net sugar transporting steps (and for the exchange reaction). The free energy of the putative rate-limiting step in absence of ∆GraphicH+ (opening of the periplasmic cavity) is indicated by the vertical red arrow (ΔGn*). The hypothetical effect of an imposed ∆GraphicH+ is shown as a broken black vertical arrow, and the broken red lines show the resulting energy profile.
(C) Postulated steps in the transport cycle of a uniporter. Steps 1–4 are similar to steps 1–4 in A but without release of a H+. Steps 5–8 are the same as in A.
(D) Hypothetical energy profile for the transport cycle of a uniporter. The colors of the lines correspond to parts of the transport reaction equivalent to A. The energy trap for binding an inhibitor is indicated by the broken blue line.
Homology-based modelling of GLUT1 structure.
a, A structural model of GLUT1 is generated on the basis of XylE structure and sequence conservation. Invariant and conserved residues are coloured magenta and dark green, respectively. b, Predicted secondary structural elements of GLUT1. Polar and aromatic residues predicted to be involved in D-glucose binding are shaded red and yellow, respectively. The residues whose mutations were found in GLUT1 deficiency syndrome are shaded purple and blue for invariant and variant residues, respectively. c, Mapping of the disease-related residues on the structural model of GLUT1. Invariant and variant residues in XylE and GLUT1–4 are labelled red and blue, respectively. d, Mutation of Arg 133 or Arg 341 in XylE, which correspond to Arg 126 and Arg 333 of GLUT1 (highlighted in magenta in c), led to abrogation of D-xylose transport in a liposome-based counterflow assay.
The biochemical characterizations reported revealed that the SP motifs as well as the intracellular helix domain have a critical role for the function of XylE. Given that extensive hydrogen bonds are formed by these structural elements, some of the residues may contribute to proton symport. Nevertheless, it remains puzzling that all these residues are invariant in GLUT1–4 and XylE, the former being facilitative uniporters and the latter being a proton symporter
Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE 8
Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.
The major facilitator superfamily (MFS) of secondary membrane transporters is the largest collection of membrane proteins that are found in all kingdoms of life and includes >15,000 members. They function as uniporters, antiporters or symporters a to catalyse the transport of a wide range of compounds including simple sugars, inositols, drugs, amino acids, oligosaccharides, nucleosides, esters and a large variety of organic and inorganic ions. MFS members share a remarkable structural conservation. MFS proteins contain two halves, the N- and C-terminal domains, each formed by a bundle of six or seven transmembrane (TM) helices and the substrate translocation pathway is located at the interface between the N- and C-domains
(a) The starting outward partially occluded conformation with D-xylose bound,
(b) the occluded intermediate,
(c) the target inward open conformation and
(d) the final sugar release conformation of XylE embedded in the membrane system in the MD simulation box. XylE (van der Waals surface) is embedded in the lipid molecules (yellow and orange lines) surrounded by water molecules (light-blue spheres) and Cl− ions (magenta spheres). D-xylose molecule is shown as white and red large spheres.
a A symporter is an integral membrane protein that is involved in the transport of many differing types of molecules across the cell membrane. The symporter works in the plasma membrane and molecules are transported across the cell membrane at the same time, and is, therefore, a type of cotransporter.