Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Jia, Ruyu; Martens, Chloé; Shekhar, Mrinal; Pant, Shashank; Pellowe, Grant A.; Lau, Andy M.; Findlay, Heather E.; Harris, Nicola J.; Tajkhorshid, Emad; Booth, Paula J.

doi:10.1101/2020.04.03.023564

Cited by 12 publications

(14 citation statements)

References 41 publications

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“…7). These findings are also in agreement with a recent molecular dynamics study revealing that a D-glucose molecule within the XylE-binding pocket has a lower degree of motion compared with D-xylose (66).…”

Section: D-glucose Acts As An Inhibitor For Xyle By Interactions With...supporting

confidence: 92%

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bazzone

Tesmer

Kurt

et al. 2022

Journal of Biological Chemistry

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Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine K M , V max , K D , and k obs values for different sugar substrates. K D and k obs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H + symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H + and d -glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s −1 ) and for sugar translocation (2 s −1 − 30 s −1 for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d -glucose can act as an inhibitor for XylE.

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Section: D-glucose Acts As An Inhibitor For Xyle By Interactions With...supporting

confidence: 92%

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bazzone

Tesmer

Kurt

et al. 2022

Journal of Biological Chemistry

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“…Unlike higher resolution approaches, HDX-MS offers the advantage of obtaining dynamic structural information for samples that presents heterogeneous and/or flexible areas. It was successfully applied to characterize the dynamics and interactions of membrane transporters and even GPCRs (48)(49)(50)(51)(52)(53).…”

Section: Hdx-ms Gives Information Related To Solvent Accessibility and Dynamics Of Biological Complexes It Is Based On The Exchange Kinetmentioning

confidence: 99%

Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus

Grison

Lambey

Jeannot

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Atypical chemokine receptor 1 (ACKR1) is a G protein–coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein–binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1−Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

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“…For HDX, the application of the standard HDX MS protocols to MPs has not been straightforward [26,29–34]. In recent years, however, there were a few MS‐based HDX studies on MPs [35–37]. For example, by combining HDX MS and molecular dynamics (MD), the conformational dynamics of secondary transporters at the molecular level was revealed [35].…”

Section: Introductionmentioning

confidence: 99%

“…The authors show that HDX measurements in nanodiscs of different lipid compositions can be used to understand how specific lipid environments fine‐tune the conformational dynamics of transporters. They also combined HDX MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter, XylE, in micelles and demonstrated the ability of HDX MS to distinguish conformational dynamics of inhibitor and substrate binding in very recent work [37].…”

Section: Introductionmentioning

confidence: 99%

Advances in mass spectrometry‐based footprinting of membrane proteins

Gross

2022

Proteomics

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Structural biology is entering an exciting time where many new high‐resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo‐electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS‐based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

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Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Cited by 12 publications

References 41 publications

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus

Advances in mass spectrometry‐based footprinting of membrane proteins

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