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

Abstract

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-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.

Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS)

“…Given its salt bridge interaction with Arg133, the Asp27 must be in a deprotonated state, consistent with MD simulations. 272 Yet, the fact that the substrate can bind in this conformation implies that protonation of Asp27 is not a prerequisite for substrate binding in XylE, unlike in LacY and other H + -coupled symporters, where protonation of a key residue in the substrate binding site is required in the wild-type situation. 90 In contrast to LacY, the transporter XylE, like many of the monosaccharide sugar porters, lacks an ionizable residue in the sugar binding pocket ( Figure 10 b).…”

“…Consistent with this rationale, recent HDX-MS and MD simulations of XylE concluded that (i) when Asp27 is negatively charged, the binding of d -xylose stabilizes the outward-facing state and is stably bound, (ii) upon Asp27 protonation, TM1 become more dynamic and increased water solvation that leads to d -xylose adopting multiple states, and (iii) that Asp27 protonation is a critical switch for conformational transition into the inward-facing state. 272 …”

“… 132 The potential explanation for this behavior is that GlcP Se lacks a nearby glutamate residue that can further fine-tune the p K a of the TM1 aspartate as it does in XylE. 132 , 272 Though unclear, the data nonetheless implies that the energetic differences between H + bound and unbound events can be low enough that H + and sugar translocation are not always strictly coupled. Indeed, just as mutants can be found that abolish the requirement for H + -coupling, other mutants can also lead to H + -leaks as shown in LacY.…”

General method to stabilize mesophilic proteins in hyperthermal water

“…The structure-function relationship of amphiphilic polymers and their thermal stabilization effect was investigated. All-atom classical molecular dynamics (MD) simulations of complexes (BSA with and without PEG or/and C 18 fragment, respectively) were performed in Amber18 package to study the stability of protein backbone under the different temperatures ( Bondanza et al., 2020 ; Jia et al., 2020 ). Final 100 ns trajectories were used for further analysis.…”

Mechanisms of GPCR hijacking byStaphylococcus aureus

“…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 (Du et al, 2019;Fiorentino et al, 2020;Jia et al, 2020;Möller et al, 2019;Reading et al, 2020;Zhang et al, 2010). HDX-MS optimization for ACKR1 allowed the identification of 77 peptides covering 70.1 % of WT ACKR1, with 3.41 peptide redundancy (Fig.…”