%0 Journal Article
%T Engineered XylE as a tool for mechanistic investigation and ligand discovery of the glucose transporters GLUTs
%A Jason Ye Lin
%A Jianping Wu
%A Meng Ke
%A Nieng Yan
%A Shuo Zhang
%A Xin Jiang
%A Yafei Yuan
%J Archive of "Cell Discovery".
%D 2019
%R 10.1038/s41421-019-0082-1
%X a Structure-guided introduction of disulfide bonds to lock XylE in constitutively inward-facing (upper) and outward-facing (lower) conformations. ExoCC: disulfide bond on the extracellular or exofacial side, EndoCC: disulfide bond on the intracellular or endofacial side. In the cartoon diagram, the cysteine mutations for disulfide bond formation are highlighted and a DrICE protease cutting site, indicated by the red scissor in each panel, was introduced for validation of the disulfide-bond formation. In the absence of the indicated disulfide bond, DrICE would cleave both XylE variants to two segments that can be separated on reducing SDS-PAGE. After formation of disulfide bond, the XylE variants would remain to be one band on SDS-PAGE after DrICE cleavage. b Biochemical validation of disulfide bond formation. The ExoCC and EndoCC XylE variants were both purified to homogeneity and subjected to DrICE proteolysis under indicated conditions. The distinct patterns on reducing SDS-PAGE with or without reducing agent DTT confirms the formation of designed disulfide bonds. c Summary of ITC measurement of the binding affinity between xylose and XylE variants. d The XylE-WW lost transport activity. Control group refers to protein-free liposome in the counterflow assay. The transport activity of XylE-WW is normalized relative to that of wild-type (WT) XylE. Error bars represent s.d. e The crystal structure of XylE-WW displays an outward-facing conformation. The corresponding TM segments in the four 3-helix repeats in a MFS fold are colored the same, pale purple for TMs 1/4/7/10, pale cyan for TMs 2/5/8/11, and green for TMs 3/6/9/12. The extracellular and intracellular helices are colored marine and orange, respectively. The two introduced Trp residues are shown as magenta sticks. f Minor conformational changes between the outward-facing structures of WT XylE and XylE-WW. Left: overall structure comparison between XylE-WW (green) and wild type XylE (gray). XylE-WW structure superposes with wild type XylE structure with r.m.s.d 0.794£¿£¿. Right: structure alignment of N/C domain between XylE-WW and wild-type XylE. Wild-type XylE are colored gray. N domain of XylE-WW is colored green. C domain of XylE-WW is colored marine. The N-domain (residues 5¨C219) and C-domain (residues 277¨C462) superpose with wild type XylE with r.m.s.d 0.642 and 0.583£¿£¿, respectively. g Outward-facing XylE has higher affinity for d-xylose and d-glucose than WT XylE. The affinities between the XylE variants and the ligands were measured by Microscale thermophoresis analysis (MTS). h The
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399220/