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Review
. 2023 Mar 29;67(2):255-267.
doi: 10.1042/EBC20220190.

Structural mass spectrometry approaches to understand multidrug efflux systems

Benjamin Russell Lewis  1 , Ryan Lawrence  1 , Dietmar Hammerschmid  1 , Eamonn Reading  1
Affiliations
Review

Structural mass spectrometry approaches to understand multidrug efflux systems

Benjamin Russell Lewis et al. Essays Biochem. .

Abstract

Multidrug efflux pumps are ubiquitous across both eukaryotes and prokaryotes, and have major implications in antimicrobial and multidrug resistance. They reside within cellular membranes and have proven difficult to study owing to their hydrophobic character and relationship with their compositionally complex lipid environment. Advances in structural mass spectrometry (MS) techniques have made it possible to study these systems to elucidate critical information on their structure-function relationships. For example, MS techniques can report on protein structural dynamics, stoichiometry, connectivity, solvent accessibility, and binding interactions with ligands, lipids, and other proteins. This information proving powerful when used in conjunction with complementary structural biology methods and molecular dynamics (MD) simulations. In the present review, aimed at those not experts in MS techniques, we report on the current uses of MS in studying multidrug efflux systems, practical considerations to consider, and the future direction of the field. In the first section, we highlight the importance of studying multidrug efflux proteins, and introduce a range of different MS techniques and explain what information they yield. In the second section, we review recent studies that have utilised MS techniques to study and characterise a range of different multidrug efflux systems.

Keywords: antimicrobial resistance; drug efflux; mass spectrometry; membrane proteins.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. Families of MDR efflux pumps
This schematic delineates the efflux pumps found within the membranes of both bacterial and eukaryotic cells that are responsible for the acquisition of MDR, along with their associated energy-coupling mechanisms. Examples of each pump are detailed in brackets. The MFS family of transporters is demonstrated as a human symporter facilitating substrate export as observed in cancer cells [88]; however, this family can act as an export or import transporter functioning via uniport, symport, or antiport. Abbreviations: MDR, multidrug resistance; ABC, ATP-binding cassette; MATE, multidrug and toxin extrusion; MFS, major facilitator superfamily; PACE, proteobacterial antimicrobial compound efflux; RND, resistance-nodulation-cell division; SMR, small multidrug resistance.
Figure 2
Figure 2. Structural MS toolbox
Native MS preserves noncovalent protein interactions during the transition to the gas phase, and reports on subunit stoichiometry, ligand binding, and protein architecture. Hydrogen/deuterium exchange (HDX) MS involves the substitution of amide hydrogens on the peptide backbone for deuterium, reporting on protein structural dynamics. Chemical cross-linking (XL) MS requires the use of a chemical cross-linker to provide information on distance restraint and structure validation. Covalent labelling MS allows for surface mapping and reports on tertiary structure. Abbreviations: ESI, electrospray ionisation; nESI, nano-electrospray ionisation; LILBID, laser-induced liquid bead ion desorption; DESI, desorption electrospray ionisation; MS, mass spectrometry.
Figure 3
Figure 3. Chlorhexidine binding to AceI monitored by native MS
(A) Structures of AceI as predicted by AlphaFold [89,90] and chlorhexidine. (B) Mass spectra of 5 μM AceI with increasing amounts of chlorhexidine, from 0 to 40 μM. AceI presents as a mixture of monomers and dimers. Satellite peaks next to the main charge state distribution of AceI correspond to the mass of chlorhexidine, and hence represent AceI-chlorhexidine complexes. Chlorhexdine increases the proportion of AceI dimers in the mass spectrum. Adapted with permission from Bolla et al. [57].
Figure 4
Figure 4. Mapping the binding sites of AcrA and TolC to PG with XL-LC/MS-MS
(A) The crystal structure of AcrA (PDB: 2F1M), with the yellow areas representing peptides that interact with PG. Insert displays how DTSSP reacts with primary amines to create the cross-linked product. Lysine residues which can react with DTSSP have their side chains shown. (B) The crystal structure of TolC (PDB: 1EK9), with the yellow areas representing peptides that interact with PG. Only one protomer in the TolC trimer is coloured for clarity, and the mapped peptides can be seen more clearly in the zoomed in insert. Adapted from Shi et al. with permission [91]. Abbreviations: XL, cross-linking; LC/MS-MS, liquid chromatography tandem mass spectrometry.
Figure 5
Figure 5. The effect of cholesterol on the structural dynamics of P-gp
(A) Structure of P-gp (PDB: 5KPI) with the differential hydrogen deuterium exchange (ΔHDX) painted on. ΔHDX = P-gp cholesterol nanodiscs – P-gp DMPC only nanodiscs. Deprotected peptides are coloured red, protected peptides are coloured blue, areas with insignificant ΔHDX are coloured yellow, and regions not covered are grey. Only peptides with significance across two or more timepoints were deemed ‘affected’ (95% significance). The greyscale structure at the bottom represents the two halves of P-gp: white = residues 1–650 and grey = residues 651–1284. (B) Woods plot of the ΔHDX data across all timepoints and the sum of all timepoints. The deuterium uptake for the peptides is shown on the Y-axis and the sequence of the peptides shown on the X-axis. The domains of the corresponding peptides are labelled at the top. (C) Peptide uptake plots for four peptides across different domains. Peptide uptake plots show percentage deuteration as a function of time for a given peptide. Obtained with permission from Biochemistry [75].
Figure 6
Figure 6. The increasing complexity of studying efflux protein systems
Schematic showing the different environments used to study membrane proteins, with increasing complexity and representability.
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