Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190914_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190419_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190617_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190609_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190629.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190624.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190606.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190607_PLGSOUTPUT.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190615.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190411.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190622_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190510_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190411_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190916.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190504.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190916_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190615_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190609.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190914.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190207.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190426_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190624_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190419.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190629_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190207_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/190504_plgsoutput.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190621.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190617.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190426.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/03/PXD018145/20190510.zipprimaryOK200argyris.politis@kcl.ac.ukRuyu JiaHDMSEMass SpectrometryNot availableSecondary transportersHydrogen-deuterium exchange mass spectrometry (hdx-ms)Molecular transportConformational dynamicsMajor facilitator superfamilyMolecular dynamics simulationsMembrane proteinshttp://www.ebi.ac.uk/pride/archive/projects/PXD018145Cell CultureXylE was overexpressed in E.coli BL21-AI (Invitrogen), which was transformed with the XylE gene in the presence or absence of the chosen mutations and cloned in the (30ug/ml) kanamycin-resistant Pet28 plasmid (Novagen) modified with a C-terminal 10-histidine tag, grown in 6 baffled flasks each containing 2L of LB media at 37 ℃ 220 r.p.m to an OD600 of 0.8. Expression was induced with 1mM IPTG (Isopropy-b-D-1-thiogalactopyranoside) and 0.1% (w/v) arabinose and growth continued until the value of OD600 is flat. The cells were harvested by centrifugation for 30mins at 4,200 r.p.m, and washed in 200ml PBS buffer and centrifuged again for 20mins at 4,200 r.p.m. The pellet was then resuspended in 50ml PBS (phosphate-buffered saline) with10mM β-mercaptoethanol and 1 cOmplete protease inhibitor tablet and was frozen at -70℃ before purification. Cells were defrosted and incubated with 1.5 µL benzonase nuclease (ThermoFisher) for 10 mins at room temperature before passed through constant cell disrupter at 25 kPsi, 4℃. Then the ice-chilled membranes were isolated by ultracentrifugation for 30min at 38,000 r.p.m, 4℃. Membrane pellets were solubilised for 2 hours with mixing in solubilisation buffer [50mM sodium phosphate pH 7.4, 200mM NaCl, 10% (v/v) glycerol, 20mM imidazole, 10mM β-mercaptoethanol, and 2% β-DDM (Anatrace), 0.1mM phenylmethylsulfonyl fluoride (PMSF) and EDTA free protease inhibitor tablet (Roche)] in the cold room (4℃). Then the protein solution was isolated by centrifugation for another 30mins at 38,000 r.p.m to remove DDM insoluble material. The supernatant was filtered using 0.22µm filter before injected into the AKTA in cold room. The filtered protein solution was first applied to a Ni-NTA column equilibrated in 96% SEC purification buffer [50mM sodium phosphate pH 7.4, 10% (v/v) glycerol, 2mM β-mercaptoethanol, and 0.05% β-DDM (Anatrace), 0.1mM phenylmethylsulfonyl fluoride] and 4% elution buffer [50mM sodium phosphate pH 7.4, 500mM imidazole, 10% (v/v) glycerol, 10mM β-mercaptoethanol, 0.1mM phenylmethylsulfonyl fluoride (PMSF) and 0.05% β-DDM (Anatrace)]. Then protein was washed with 50ml 85% SEC purification buffer, 15% elution buffer and only 2ml eluted fraction passed through 100% elution buffer was collected for further size exclusion chromatography (SEC) purification program. The SEC purification was conducted with a Superdex 16/600 GL SEC column, which was equilibrated with 1.2 column volume SEC purification buffer. The elution fraction was collected and concentrated with viva spin concentrator (100 kDa cutoff). The samples were either flash frozen and kept at -70 ℃ until use or used directly for HDX-MS experiments.PrideNot availableNo PTMs are included in the datasetAcquired reference MSE data was put on PLGS (ProteinLynx Global Server 2.5.1, Waters) to achieve the peptide map, then all the MSE data including reference and deuterated samples were processed by DynamX v.3.0 (Waters) for deuterium uptake determination. Peptide filtration and analysis were performed as described before. Woods plots were generated by the in-house Deuteros software.ProteomicsArgyris PolitisSynapt MSPARTIALChemistry Department, King's College LondonEscherichia Coli33268777 Jia R, Martens C, Shekhar M, Pant S, Pellowe GA, Lau AM, Findlay HE, Harris NJ, Tajkhorshid E, Booth PJ, Politis A. Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter. Nat Commun. 2020 11(1):6162 10.1038/s41467-020-20032-3ruyu.jia@kcl.ac.ukKing's College LondonUnited KingdomProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter.Jia Ruyu R, Martens Chloe C, Shekhar Mrinal M, Pant Shashank S, Pellowe Grant A GA, Lau Andy M AM, Findlay Heather E HE, Harris Nicola J NJ, Tajkhorshid Emad E, Booth Paula J PJ, Politis Argyris Asodium salt, AU019543, 2610315D21Rik, IL1BC, CASP-1, Ethylenediaminetetraacetic acid, growth and development, chronic obstructive pulmonary disease (COPD), L-Histidine, (ethylenedinitrilo)tetraacetic acid, l(2)k09905, Mutations, dmTAF[[II]]230, protein polypeptide chains, T22F8_160, tribasic, ATGPR7, FLASH, imidazole monohydrochloride, Chronic irreversible airway obstruction, SEC, 1, 2, GRP8, GRP7, RAFT1, Il1bc, insoluble, 3-Propanetriol, DmelCG8428, Oelsuess, Spin, Kanamycin A, sarcoma of breast, TFIID TAF250, cel, N, sec, Tissue, Fluoride, CAFL - Chronic airflow limitation, proteins, RIP25, IL-1BC, Benzenemethanesulfonyl Fluoride, salt, SPIN, dibasic, column, disodium acid phosphate, disease (COPD), BTKAP1, GTFII-I, acidum edeticum, 6030441I21Rik, trisodium phosphate dodecahydrate, Glyceritol, histidina, dTAF[[II]]230, chronic obstructive lung disease [Ambiguous], Caspase-1 subunit p10, BZRP, TAF200, 2'', integral to membrane, breast sarcoma, BAP135, PBS, DmelCG7788, PBR, dibasic (anhydrous), CHRONIC OBSTRUCTIVE AIRWAY DIS, common salt, trisodium phosphate, BAP-135, O-3-amino-3-deoxy-alpha-D-glucopyranosyl-(1-6)-O-(6-amino-6-deoxy-alpha-D-glucopyranosyl-(1-4))-2-deoxy-, Airflow Obstruction, GLYCINE-RICH PROTEIN 8, Chronic Obstructive Airways Disease, Chronic obstructive lung disease, Gro, Sez7, glycerine, disodium salt, INSDC_feature:gene, N'-1, GtfII-I, L isomer, Painful bladder syndrome, l(2)W5, Bip, F2G1.4, Taf250, OBSTRUCTIVE PULMONARY DISEASE (COPD), Drice, DRICE, TAF230, BPBS, 2-ME, GLYCEROL, sarcoma of the breast, mBiP, DrIce, DrICE, number, CAO - Chronic airflow obstruction, CHRONIC OBSTRUCTIVE PULM DIS, Chronic obstructive pulmonary disease NOS, protein-containing complex, EDTA tetraanion, and RNA binding 1, Chronic obstructive pulmonary disease finding, glycyl alcohol, PULMONARY DISEASE (COPD), D2Wsu141e, AL022860, H2im(+), T6K12_14, dTAF[[II]]250, 2'''-(ethane-1, sodium (2:3) salt, 2-Ethane diylbis-(N-(carboxymethyl)glycine), cell, Tissues, ethylenediamine tetraacetic acid tetraanion, absent from organism, Glycerin, PCE-2, Histidin, pk18, MBR, Solution, dTAF250, glycerolum, Caspase-1 subunit p20, ATGRP7, ATGRP8, NEC, Kochsalz, PTBR, Chronic, Frap1, Chromatographies, dibasic sodium phosphate, Al-imidazole, histidine, WBSCR6, constant, L-isomer Histidine, chronic obstructive airways disease NOS, caspase 3, COAD, FRAP2, BG:DS00004.13, CE-2, COLD - Chronic obstructive lung disease, buffer, Cell, chronic, bnch, dTAF230, l(2)k02511, PBS buffer, Temperatures, native protein, Natriumchlorid, TAF[[II]]250/230, (COPD), chronic airway obstruction, NaCl, chronic obstructive, CAL - Chronic airflow limitation, T6K12.14, Taf[[II]]250, covalent modifier, postnatal growth, GR-RBP7, GR-RBP8, IL-1 beta-converting enzyme, Interleukin-1 beta-converting enzyme, imidazolium, FRAP, mDRC, monobasic anhydrous, disodium hydrogen phosphate, chronic obstructive lung disease (disorder), dodecahydrate, 2-mercapto-, CXorf43, quantitative, phosphates, Benzenemethanesulfonyl, ethylenediaminetetraacetate tetraanion, chronic obstructive pulmonary disease, DIWS, imidazole acetate, T22F8.160, insensitive, and rna binding 2, Chronic Airflow Obstructions, postnatal development, CG7788, crice, protein, Pi, Membrane Tissues, Ethanol, halite, Glycerol, D030011N01Rik, chronic obstructive airways disease NOS (disorder), Phenylmethylsulfonyl, DL-Histidine, Williams-Beuren syndrome chromosomal region 6 protein, 2', Chronic Obstructive Pulmonary Disease (COPD), protein aggregate, Edetic acid, 2-diyldinitrilo)tetraacetate, COLD (chronic obstructive lung disease), ion(4-), kDa, NEC in ICD9CM_2006, membrane region, 2 Mercaptoethanol, Kantrex, IBP, Tablet, free, Chronic airflow limitation, cloruro sodico, mKIAA1315, Gm380., ICE, GLYCINE RICH PROTEIN 7, 2-amino-3-(1H-imidazol-4-yl)propanoic acid, Diws1t, L-Arabinose, sodium hydrophosphate, (ethane-1, Bladder pain syndrome, sodium biphosphate, Grp78, glycine-rich RNA-binding protein 8, CHRONIC OBSTRUCTIVE PULMONARY DISEASE, CHRONIC, Membrane Tissue, monobasic, ethylenediaminetetraacetate, rock salt, obstructive lung disease, ice, iCE, drice, 32P-labeled, D2Wsu17e, {[-(BIS-CARBOXYMETHYL-AMINO)-ETHYL]-CARBOXYMETHYL-AMINO}-ACETIC ACID, TAFII-250, membranous organ component, CED-4, TAF250/230, Airflow Obstructions, Programs, pulmonary disease (COPD), alpha-Amino-1H-imidazole-4-propionic acid, TAFII250, imidazole conjugate monoacid, AA387232, anhydrous, drIce, drICE, low temperature, CHRONIC OBSTRUCTIVE, glycerol, Kanamycin Sulfate, chronic obstructive pulmonary disease and allied conditions, SRF-Phox1-interacting protein, Chronic airway disease, NOS, phosphoric acid, RAPT1, COPD NOS, CHRONIC OBSTRUCTIVE LUNG DIS, Chronic airway obstruction, l(2R)W5, absence, Painful Bladder Syndrome, membrane of organ, resistant, growth pattern, cold, non-developmental growth, P45, L-isomer, L Arabinose, CG17603, natrii chloridum, TAF[[II]], monosodium salt, Chronic Airflow Obstruction, imidazolium chloride, phosphate, WBS, phosphate ions, SR3-5, COPD - Chronic obstructive pulmonary disease, 2-Mercaptoethanol, chronic obstructive airways disease, p45, CG8428, imidazolium ion, obstructive pulmonary disease (COPD), COLD, chronic obstructive lung disease, IMIDAZOLE, Bladder Pain Syndrome, sodium dihydrogen orthophosphate, d230, DISEASE (COPD), Histidine, D4Ertd659e, dTAFII250, ethylenediaminetetraacetic acid, circadian rhythm, baffled, disodium hydrogen phosphate anhydrous, EfW1, neutral sodium hydrogen phosphate, presence, TFII-I, Buffer, Bruton tyrosine kinase-associated protein 135, Glycerine, Cold, Propanetriol, Pulmonary Disease, polypeptide chain, dmTAF1, Taf230, Phenylmethanesulfonyl Fluoride, integral component of membrane, Acide ethylenediaminetetracetique, Trihydroxypropane, sodium dihydrogen phosphate, imidazole citrate, IB291, Hsce70, AA673430, acide edetique, TAF250, SEZ-7, Taf200, Cold Temperatures, plate-like, D-Streptamine, propane-1, Selb, uniform, chlorure de sodium, Taf1p, flat, Chronic Obstructive, edta, Benzenemethanesulfonyl fluoride, 2-diyldinitrilo)tetraacetic acid, EDTA, region of membrane, Cu-imidazole, BTK-associated protein 135, AA553322, TAF, imidazole sodium, TAG, imidazole monophosphonate, COPD, membrane, COAD - Chronic obstructive airways disease, TAF[[II]]250, heptahydrate, sodium phosphate, DBI, protein complex, Glyzerin, not elsewhere classified, 3-triol, l(3)84Ab, tag, PULM DIS CHRONIC OBSTRUCTIVE, acido edetico, Chronic obstructive pulmonary disease finding (finding), development, count in organism, secret agent, PKBS, natural protein, p230, Protein, whole membrane, TFIID, table salt, 25/11, CES2A1, GLYCINE-RICH RNA-BINDING PROTEIN 7, 1H-imidazol-3-ium, Phenylmethanesulfonyl, sodium phosphate monobasic anhydrous, trisodium salt, chronic obstructive airway disease, 3-Trihydroxypropane, Temperature, transmembrane, TAF[[II]]230, Dops, H4edta, AI327068, TAF[II]250, Membrane, Interleukin-1 beta convertase, l(2)10403, DmelCG17603, monosodium dihydrogen phosphate, 3.4.22.36, cold (chronic obstructive lung disease), edetic acid, Chronic Obstructive Lung Disease, growth, presence or absence in organism, TAF1, sodium chlorideMass Spectrum Analysis, inhibitors, Mass Spectrum, H, distinct, Analyses, hydrogen, Wasserstoff, Spectrometry, Hydrogen 2, Deuterons, inhibiteur, inhibitor, Protium, Spectrum Analyses, Spectrum Analysis, Spectroscopy, Hydrogen-2, Hydrogen-1, ligand., inhibidor, Mass, hidrogeno, Analysis, 1H, hydrogene, Mass Spectrum Analyses, Mass SpectroscopyApplications Software, MUTYH-related attenuated familial adenomatous polyposis, type 2, Open Source, BBE, data, MUTYH-Associated Polyposis, Computer Software, methionine aminopeptidase activity, determination, AI447866, MYH-associated polyposis, 2-phospho-D-glycerate hydro-lyase, autosomal recessive familial adenomatous polyposis, Eno-3, Maps, Computer, not genetically inherited, Peptide, Source Softwares, Software Tools, Tool, Programs, polypeptide, peptide, Polypeptides, Program, Computer Applications, uptake, Software Tool, ClvPrd, peptido, Filtrations, MUTYH-related attenuated FAP, Plgs, chemical analysis, Computer Applications Software, Computer Applications Softwares, 2, Software Engineering, Softwares, Woods, Software, familial adenomatous polyposis, Computer Program, MAP, peptidase M activity, Application, 4.2.1.11, autosomal recessive, adenomas, MUTYH-associated polyposis, MUTYH-related AFAP, Open Source Softwares, RUTBC3, rabGAPLP, L-methionine aminopeptidase activity, Software Applications, peptides, Skeletal muscle enolase, Source Software, Software Application, familial adenomatous polyposis 2, Open, Engineering, Hydrogen 2, Deuterons, Open Source Software, Computer Programs and Programming, RabGAP-5, Muscle-specific enolase, MYH-Associated Polyposis, MSE, RABGAP5, Computer Software Application, autosomal recessive multiple colorectal adenomas, Computer Programs, Hydrogen-2, Applications, 1110030K05Rik, Applications Softwares, Tools, RUSC3, Computer Software Applications., multiple colorectal, Peptid, peptidos, MAP syndrome, MUTYH-related attenuated familial polyposis coli, GSD13, Polypeptide, assay, FAP2, Enolase 3, colorectal adenomatous polyposisNetworks, gluco-hexose, obv, type 1, sugar:proton symporter activity, Family Member, Kinship, GRP1/cytohesin 1, experimental, Glucose, Glukose, myxoid malignant fibrous histiocytoma, DmelCG9819, myxoid MFH, D-Xylose, number, CG4832, Monohydrate, sugar transporter, Visible Light, Network, (DL)-Isomer, presence, supernumerary, dIKK-gamma, Solvent, Ly113, CG11633, cytohesin/GRP1, solute:solute exchange, D030011N01Rik, DmIKK-gamma, GRP1, myxoid fibrous histiocytoma, Dextrose, hnu, Grp1, symptoms, Life Cycle, light, dmIKKgamma, xylose, IKK[[gamma]], foton, sugar porter activity, IKKg, Family Life Cycle, KEY, Key, proton:sugar symporter activity, mat(2)syn-C, CnnA14D, CNN, Cnn, Marfan's syndrome, increased, methods, Kinship Network, Research, cen, Lichtquant, experimental section, ligand, PTPSTEP, inhibiteur, l(2)SH2 0323, Proton, Visible, CG 4832, DL-xylose, carbohydrate transporter activity, DL-glucose, Motions, glucose, reaction, inhibidor, Neural-specific protein-tyrosine phosphatase, IKK, Xyl, l(2)k08110, Cnn1, photon, carbohydrate-importing ATPase activity, TR2, PP2B, Kinship Networks, Marfan syndrome type 1, Family Life Cycles, Family, Anhydrous, fibromyxosarcoma, gamma, GPH, cation/sugar symporter activity, inhibitors, screening, Ligand, single-organism transport., lactose/glucose efflux transporter activity, Radiation, findings, canA-14F, D27, Family Research, (beta-D)-Isomer, Marfan syndrome, D Xylose, inhibitor, CnnA14D(I), stepk, Light, CD258, Family Members, IKKgamma, DmIKKgamma, count in organism, DmelCG4832, LIGHT, Ions, single-organism transport, dIKK, Kenny, 3.1.3.48, Xylose, Mutageneses, MFS1, (alpha-D)-Isomer, Filiation, l(2)SH0323, Hydrogen Ions, Hydrogen Ion, CG9819, sugar:hydrogen symporter activity, sugar efflux transmembrane transporter activity, Gm380, Visible Radiations, Radiations, CYH1, D-Glucose, Hydrogen, Visible Radiation, carbohydrate uptake transporter activity, myxofibrosarcoma, HVEML, Step, CG11628, HVEM-L, Dmikkgamma, increased number, Photoradiation, IKK-gamma, sugar:hydrogen ion symporter activity, mat(2)synHK21, signs, hydrogen:sugar symporter activity, Striatum-enriched protein-tyrosine phosphatase, CG18370, CG16910, MFS, hydrogen:sugar transporter activity, Anhydrous Dextrose, LTg, DmCanA-14F, DmelCG11628, experimental procedures, Life Cycles, present in greater numbers in organism, light quantum, Photoradiations, DmelCG16910, Ion, D Glucose, STEP, Families, xylo-pentose, CXorf43, small molecule transport, PpD27, quantitative, Glucose Monohydrate, Relatives, mfs, accessory, Glc, presence or absence in organismgluco-hexose, d230, GRP1/cytohesin 1, Glucose, Glukose, number, D-Xylose, Monohydrate, dTAFII250, (DL)-Isomer, EfW1, presence, dIKK-gamma, CG11633, dmTAF[[II]]230, cytohesin/GRP1, solute:solute exchange, dmTAF1, Taf230, D030011N01Rik, integral component of membrane, DmIKK-gamma, GRP1, Dextrose, Grp1, dmIKKgamma, xylose, IKK[[gamma]], Nutrient, IKKg, KEY, Key, TAF250, Taf200, dTAF[[II]]250, TFIID TAF250, cel, cell, ligand, nutrients, PTPSTEP, membrane region, inhibiteur, l(2)SH2 0323, aspartate(2-), Taf1p, Proton, DL-xylose, DL-glucose, dTAF250, glucose, reaction, inhibidor, Neural-specific protein-tyrosine phosphatase, IKK, Xyl, l(2)k08110, region of membrane, associated, TAF, Anhydrous, Macronutrient, GPH, inhibitors, nutrient, dTAF[[II]]230, TAF[[II]]250, Ligand, single-organism transport., (beta-D)-Isomer, D Xylose, TAF200, integral to membrane, l(3)84Ab, inhibitor, stepk, BG:DS00004.13, TAFII-250, TAF250/230, aspartate, Cell, dTAF230, Macronutrients, IKKgamma, count in organism, DmIKKgamma, TAFII250, Ions, single-organism transport, dIKK, p230, Kenny, 3.1.3.48, whole membrane, Mutageneses, Xylose, TAF[[II]]250/230, TFIID, aspartic acid dianion, (alpha-D)-Isomer, l(2)SH0323, Hydrogen Ions, Hydrogen Ion, Gm380, 2-aminosuccinate, CYH1, Taf[[II]]250, D-Glucose, Hydrogen, transmembrane, TAF[[II]]230, Step, CG11628, Dmikkgamma, 2-aminobutanedioate, IKK-gamma, TAF[II]250, Striatum-enriched protein-tyrosine phosphatase, CG17603, CG16910, TAF[[II]], Anhydrous Dextrose, DmelCG11628, DmelCG17603, DmelCG16910, Ion, Taf250, D Glucose, STEP, SR3-5, xylo-pentose, CXorf43, small molecule transport, quantitative, Glucose Monohydrate, TAF230, TAF1, presence or absence in organism, Glcinhibitors, metastatic., inhibiteur, inhibitor, inhibidor, distinct, ligandfalseDistinct dynamics upon substrate and inhibitor binding to a secondary transporterThe bacterial sugar transporter XylE is a proton-coupled symporter from the Major Facilitator Family (MFS) that has been crystalized in multiple conformations, and with both substrate xylose and inhibitor glucose bound. However, this series of static pictures does not capture the coupling between ligand binding and conformational changes required for transport. Furthermore, the ability for the transporter to discriminate between substrate and inhibitor is puzzling in light of the similarity of the bound structures. Here, we combine differential HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of transport in XylE. We show that protonation of D27 is a key event for conformational transition from outward-facing (OF) to inward-facing (IF). Strikingly, this transition only occurs in the presence of substrate xylose, while inhibitor glucose locks the transporter in the OF state. We corroborate our experimental findings with MD simulations by showing that D27 protonation in the presence of xylose, but not glucose, leads to unstable substrate binding, increased solvent accessibility, and correlated motions between the N- and C- lobes of the transporter, effectively setting the transporter up for the conformational transition. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding to XylE and propose that the allosteric coupling between ligand binding and protonation is a key step to initiate transport.2021-03-232020-03-23PXD0181454466449917558258038804333029535851114512067811080612461962461972842188090320637522995811363350906836351524260275838538791187947398007186801NCBITaxon:1280572307111430118726355557146835145262364915084727467073641135461388558218035823290313888691521677868895399784475187043511232941262469422296928123977133118099103430436548135833346079251981836592198131432059655819476214936329572637371922226938NCBITaxon:1272112478186613283882813955843451358672217554372949140816918045416193413903634530586112041623264460711210411706127443210859561496367469412831666943694559729452811320810039237514633087936944784475174206542208964317447127044597413797911194669400667145458793299352932010198126469041066114575512165994048339251216597216595160621213312831282128033952321832172920435315227794428112454349825741948147871720309498019752555536231439841356742951053948824261914548140479NCBITaxon:21575515343989903712285775975320596876231935163098005888455818178081736309431796989361604885661409859566416242515059071187021187053204963063863235554320466322261866331299456586068845351216129567171360441001602358700264203694569410167634102107612773511316582150480227933428146100293578617463313252911187002157641809699745774449156666754760445246767561917265181301941119294396336641136336721751117645102398709NCBITaxon:40559325823590927883965264982174982163472565691569387224982116544872056971666905108944835525663661410NCBITaxon:493287155689175854310245980810894447574171000589452680873228753531156126373006930391141554219325552187268723872487298727123598755529NCBITaxon:475154762422314146547730551800665564841581419198822445299767446982510216998231174673897641041051155744689444474153NCBITaxon:50557960167767446851000364875296006534911822637082485480557841299114578458359741276473194439137086326547876569835566369838192011164034624136810893161001428099612NCBITaxon:548681NCBITaxon:5820546414307747946409860995865228363737919662729058766896059606157295137221875944670875622708617110133548330903497412982938783267566430615127936543194747033424947298596108961588655974649987039202111072878272154078211908503757075298352061894804189853502471587796273002515630411902528674636046147964696443759391462472746128746011345062532566484526591972219638191552638588596NCBITaxon:615750521105172375059576131302409263419610526419407722774694163481123869497515NCBITaxon:235231703612506174767927459615384005257313136862697968666299034694171344346360347515363296440421018110867310185109760635586331496161286287246200966927294351338968583551865368595968912801726071018568729411009118696141633316780781186941436183NCBITaxon:6191118698840231719475932118691443906NCBITaxon:102982100071257118387279694836419080218787870384766455347095145122578656870861822607046183260705260707NCBITaxon:780NCBITaxon:1502451516658457702921158612184520928574707856100713370565295076192126385454195NCBITaxon:30555044573915383379913998192875NCBITaxon:10239NCBITaxon:71428291268063130957044830064175158592381055524159084128108228233446900815025754122810451930174830085210977970671356225833136185509924NCBITaxon:56938268830640527796179469958334240906151015151034809NCBITaxon:631240674482428130767070876138370828167728503515819901195195337024924053608881756261583558333760212671928532177357267119350184645177299808616787917743737111049056371215348112153239576769014726647091317047571256274579031133355085962859634792491308126387121203511082332648373153214693NCBITaxon:96153832331858626079914126220029813141313493760833341311111497019281239655833321018413856959111006581237561275045426662551519788749200375036009412934971530094114903524725175014012573983649908515151438992NCBITaxon:11552374539883747445446415259311792866432359839062887051580595536119459932716011497861603293338922853422999810894535936457428412754121536024271831627713512251179948875058NCBITaxon:1040724271550277916797181673871349130627415895521010306502780291595412629158515849327159570210022633889128768913558867551293094514647920312421641332803828035192843436909398580894621080348179392118259028788929502745609906901749342725632273218903829286757221247190400727NCBITaxon:57965374571143112335235621140206411981087135547714245072295337969835552222129167141262161154293157546139622421392139077020114319338293550833292307411292211484896160106772526499975744727580240550717629925122968335366581214053576229412816078395763206672400540296543282312273771843183224659620575549922957575759284593936053307972551811354970667143673315974953502611595564442143213818492231124060721054211109697622611117957529818184319229438119496340161433479866265281046603815181044214547448500796267825356709747731751351240565050NCBITaxon:1313607699389424688795588012014887519795962141140822414082232645614219728909101412285316435186763185431520767737677764940894144242327742NCBITaxon:17732140922088931157781154756208895929793244704207559101177499067499073129899136645112273231663170187367451011610358991513284262658721036098334139269611307981336795358893164071034015303337330158191306413992530654788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