Fig. 1: GLUT transport cycle, sugar porter ligand binding and D-glucose and D-xylose interactions with XylE.

a, The sugar porter transporter cycle has six major conformations during the transition from outward-open to inward-open. The occluded state has been observed only in the malarial transporter PfHT1 and is likely to be transient in GLUT proteins. b, Sugar binding site comparison between human GLUT3 (PDB 4ZWB), plant STP10 (PDB 7AAQ), E. coli XylE (PDB 4GBZ) and P. falciparum PfHT1 (PDB 6RW3), with the respective bound carbohydrates shown as sticks, and their C1-hydroxyls accentuated as spheres for reference and TM7a-b and TM10a-b colored yellow and cyan, respectively, and protein as cartoon (TM1, TM2 and TM4 omitted for clarity). Although GLUT3 (K_M = 1.3 mM)²⁸ and STP10 (K_M = 0.007 mM)⁵⁰ transport D-glucose, PfHT1 (K_M = 0.9 mM)²⁸ can transport many different sugars in addition to D-glucose, whereas XylE cannot transport D-glucose³⁸, but binds it with the same affinity as its preferred substrate D-xylose. All residues hydrogen bonding to D-glucose (shown as sticks) are identical apart from a TM10a residue (Ala404 in PfHT1), which in GLUT3 is a glutamate. Most residues in the N-terminal bundle surrounding the D-glucose, but not binding, are highly conserved (not shown). c, The sugar binding site and coordination of D-glucose in the outward-occluded crystal structure of XylE (PDB 4GBZ), shown as in b, and insert of the overlapping D-xylose and D-glucose (sticks, colored teal and slate, respectively) binding poses. d, The sugar binding site and coordination of D-xylose in the outward-occluded crystal structure of XylE (PDB 4GBY), where the interactions are very similar compared with those in c. Figure shown as in b. e, Top: D-xylose (500 µM) STD (red) and ¹H spectra (black) in presence of XylE–GFP reconstituted into liposomes. Bottom: as above for D-glucose. f, Left: normalized STD effects observed following addition of either D-xylose or D-glucose (500 µM) to XylE–GFP reconstituted in liposomes made from E. coli versus bovine brain-fraction-seven lipids. Right: uptake of ³H-D-xylose or ¹⁴C-D-glucose by WT XylE–GFP in proteoliposomes. Error bars: mean ± s.e.m. of n = 3 independent experiments.

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