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Monosaccharides are necessary organic compounds for eukaryotic organisms, primarily used for an energy source, but also essential for building carbon skeletons, transport molecules, and for signaling. In humans, the transport of monosaccharides, and other small carbon compounds across the cell membrane is facilitated by the GLUT family of membrane proteins, of which 14 proteins are encoded by the human genome(1). GLUT proteins belong to the major facilitator superfamily of membrane proteins, which are characterized by having 12 transmembrane helices separated into two six-helix bundles. These two bundles work together to create a “rocker-switch” mechanism which opens the protein to either the extracellular space or the cytoplasm(2).
In our September 2014 newsletter we highlighted the breakthrough which was the determination of the structure of the human GLUT1 protein by the Yan Lab at Tsinghua University(3). Before the structure of GLUT1 was determined, the only structural information available about eukaryotic glucose transporters was gleaned from homologous bacterial proteins, such as E. coli LacY, determined in 2003(4), and E. coli XylE, determined in 2012(5). At the end of 2015, two new structures from the glucose transporter superfamily, GLUT3(6) and GLUT5(7), were published. Additionally, in April 2016, additional structures of GLUT1 were cocrystallized with three different inhibitors(8), furthering our understanding of these important proteins. To commemorate this remarkable achievement, we are focusing on these three discoveries.
Like the original GLUT1 structure(3), the structure of the GLUT3 transporter was determined by the Yan Lab at Tsinghua University in Beijing(6). To determine the structure of GLUT3, the protein was solubilized using DDM, followed by purification in CYMAL-6. Purified GLUT3 was then crystallized using the LCP method, using Monoolein as the lipid. In total, three structures of GLUT3 were determined, each with different substrates bound or in a different conformation. Interestingly, the 1.5 Å resolution of the glucose-bound structure (PDB: 4ZW9) shows that both the α and ß anomers of glucose are recognized by GLUT3. Combined with the information from GLUT1, the working model for how GLUT proteins transport substrate can be updated to one where the substrate binding site, TM7, and TM10 all undergo structural rearrangement during transport.
Determined by the labs of So Iwata and David Drew, the structures of the rat and bovine fructose transporter GLUT5 show the protein in two different confirmations(7). Rat GLUT5 protein was solubilized, purified, and co-crystallized with an scFv fragment in the detergent DDM. Bovine GLUT5 was solubilized using DDM, followed by purification in DDM and UDM. Well-diffracting crystals of the bovine GLUT5 protein required the addition of HEGA-10 to the well solution. Comparison of the substrate binding sites of GLUT5 to other transporters identified one amino acid (Q166) that when mutated, switched binding preference of the protein from fructose to glucose. Analysis of the rat and bovine structures allow for elucidation of the transport mechanism of GLUT5, where in addition to the “rocker-switch” mechanism of transport, conformational changes in TM7 and TM10 also play a role in “gating” the pore.
Building upon these two studies, the lab of Robert Stroud at the University of California – San Francisco, recently determined the structure of GLUT1 in complex with three different inhibitors: cytochalasin B, GLUT-i1, and GLUT-i2(8). To determine these structures, GLUT1 was solubilized using DDM, and purified and crystallized using NG. Combined, these structures advance our understanding of the glucose binding site in GLUT1 by identifying key residues in the glucose substrate binding site, as well as identifying Trp388 in TM10 as a key binding determinant for all three inhibitors. Lastly, docking experiments of these compounds with other members of the GLUT family show some differences in selectivity which is invaluable to the design of protein specific inhibitors.
Together, these new structures of the GLUT1, GLUT3, and GLUT5 proteins refine our knowledge of how necessary monosaccharides are transported, and will invaluable in designing therapeutics for the diseases these proteins are implicated in, such as, diabetes, Alzheimer’s, cancer, and obesity.
In these notable publications, n-Dodecyl-β-D-Maltopyranoside (DDM), U300 - n-Undecyl-β-D-Maltopyranoside, Anagrade (UDM), Nonyl Glucoside (β-NG), CYMAL-6, and HEGA-10 were used in solubilization, purification, and/or crystallization. At Anatrace, we’re seriously committed to developing and supplying the industry’s finest high-purity products – and equally committed to the high standards that make it possible. Our standards have made Anatrace an internationally-recognized leader in manufacturing reagents for membrane protein studies and custom chemical synthesis. And those same standards mean you’ll have the confidence to aim higher, too.
1) Mueckler, M. and Thorens, B. (2013). Mol. Aspects. Med. 34(2-3), 121-138.
2) Yan, N. (2013) Trends Biochem. Sci. 38(3), 151-159.
3) Deng, D. et al. (2014) Nature 510(7503), 121-125.
4) Abramsom, J. et al. (2003) Science 301(5633), 610-615.
5) Sun, L.et al. (2012) Nature 490(7420), 361-366.
6) Deng, D. et al. (2015) Nature 526(7573), 391-396.
7) Nomura, N. et al. (2015) Nature 526(7573), 397-401.
8) Kapoor, K. et al. (2016) PNAS 113(17), 4711-4716.