Proteomics

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Distinct dynamics upon substrate and inhibitor binding to a secondary transporter


ABSTRACT: The 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.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Escherichia coli  

TISSUE(S): Cell Culture

DISEASE(S): Not Available

SUBMITTER: Ruyu Jia  

LAB HEAD: Argyris Politis

PROVIDER: PXD018145 | Pride | 2021-03-23

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
190207_plgsoutput.zip Other
190411_plgsoutput.zip Other
190419_plgsoutput.zip Other
190426_plgsoutput.zip Other
190504_plgsoutput.zip Other
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Publications

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 A  

Nature communications 20201202 1


Proton-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-facin  ...[more]

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