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bioRxiv · 10.1101/2022.07.11.499559

Integrative HDX-MS enables quantification of the conformational landscape of the sugar transporter XylE

Jia, R.·Bradshaw, R.·Calvaresi, V.·Politis, A.
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Abstract

A yet unresolved challenge in structural biology is to quantify conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilising them for in vitro studies. To address this challenge, we present here an integrative strategy that combines hydrogen-deuterium exchange mass spectrometry (HDX-MS) with ensemble modelling. We benchmark our strategy on wild type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments and identify key lipid contacts that modulate protein conformations. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles, allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modelling to capture, accurately quantify and subsequently visualise co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors. For Table of Content Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/499559v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@cf7b35org.highwire.dtl.DTLVardef@1a1da1eorg.highwire.dtl.DTLVardef@f70627org.highwire.dtl.DTLVardef@1b52b90_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Jia, R., Bradshaw, R., Calvaresi, V., Politis, A.. 2022-07-11. Integrative HDX-MS enables quantification of the conformational landscape of the sugar transporter XylE. https://doi.org/10.1101/2022.07.11.499559

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