Biology subjects

Reading, E.

Publications and source records attributed to Reading, E..

6 recordsLinked to original sources

bioRxiv2026

LemonCatcher Acidic Pull-Down Enables Selective In-Cell Hydrogen-Deuterium Exchange Mass Spectrometry

Proteins are dynamic molecules which sensitively adapt according to their environment. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) provides unique insights into protein conformational processes. However, existing methodology cannot selectively enrich proteins post-labeling because D-to-H back exchange must be minimized by rapid processing at pH 2.3-3.0 and 0 {degrees}C, where affinity purification fails. Here, we create LemonCatcher, a protein superglue that spontaneously forms an amide bond to the LemonTag peptide under these harsh acidic and cold quench conditions, even at -20 {degrees}C. Engineering of a bead-coupled LemonCatcher purification system introduces fast and selective quench-capture HDX-MS (SelQueX) on LemonTagged fusion proteins. We demonstrate targeted measurement of protein dynamics in living bacterial cells, revealing ligand-induced conformational changes in maltose-binding protein. Moreover, probing a stalled membrane protein nascent-chain supports a role for the ribosome in maintaining partially unfolded folding intermediates. Thus, SelQueX makes possible selective characterization of protein structural dynamics within the complex cellular milieu.

biochemistry↗

bioRxiv2026

Structural dynamics underlying agonist activation of a GLP-1R-Gs precoupled complex

G-protein-coupled receptors (GPCRs) act as allosteric transmembrane signalling machines, generating distinct cellular responses depending on the conformational states induced by ligand binding. The glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR central to insulin secretion and body-weight regulation, is a key therapeutic target for obesity-associated metabolic disease. Here, we used hydrogen-deuterium exchange mass spectrometry to characterize ligand-evoked structural dynamics within a pre-coupled GLP-1R-Gs protein complex. Non-peptide agonists Chu-128 and danuglipron elicited overlapping dynamic perturbation profiles, with distinct drug-specific effects within the transmembrane bundle. In contrast, the natural GLP-1 hormone produced a weaker stabilizing effect on receptor backbone dynamics, while its inactive metabolite exerted opposing localised destabilization. Notably, both peptides uniquely modulated the highly flexible G-protein switch III loop, a key mediator of downstream signalling. These findings pinpoint areas where structural dynamics shape agonist efficacy and facilitate functional dynamics-integrated drug discovery of non-peptide agonists. SignificanceThe development of non-peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) represents a major advance in metabolic therapeutics, addressing key limitations of current peptide-based incretin therapies while enabling improved control over receptor signalling and pharmacokinetic properties. The recent FDA approval of a first-in-class small-molecule oral GLP-1R agonist, LY3502970, highlights the translational potential of this approach. However, the molecular basis by which distinct ligands modulate GLP-1R conformational dynamics and signalling remains poorly understood. Here we report how non-peptide agonists (Chu-128 and danuglipron) and endogenous GLP-1 peptide and its inactive metabolite shape the structural dynamics of a pre-coupled GLP-1R-Gs complex, revealing patterns linked to receptor activation. Our findings provide insights that guide the rational design of next-generation GLP-1R therapeutics.

biochemistry↗

bioRxiv2025

Molecular basis for multidrug efflux by an anaerobic RND transporter

Bacteria can resist antibiotics and toxic substances within demanding ecological settings, such as low oxygen, extreme acid, and during nutrient starvation. MdtEF, a proton motive force-driven efflux pump from the resistance-nodulation-cell division (RND) superfamily, is upregulated in these conditions but its molecular mechanism is unknown. Here, we report cryo-electron microscopy structures of Escherichia coli multidrug transporter MdtF within native-lipid nanodiscs, including a single-point mutant with an altered multidrug phenotype and associated substrate-bound form. We reveal that drug binding domain and channel conformational plasticity likely governs promiscuous substrate specificity, analogous to its closely related, constitutively expressed counterpart, AcrB. Whereas we discover distinct transmembrane state transitions within MdtF, which create a more engaged proton relay network, altered drug transport allostery and an acid-responsive increase in efflux efficiency. Physiologically, this provides means of xenobiotic and metabolite disposal within remodelled cell membranes that presage encounters with acid stresses, as endured in the gastrointestinal tract.

biochemistry↗

bioRxiv2024

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

biochemistry↗

bioRxiv2022

Conformational restriction shapes inhibition of a multidrug efflux adaptor protein

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and {beta} domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

biochemistry↗

bioRxiv2020

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

Resistance-nodulation-division (RND) efflux pumps play a key role in inherent and evolved multidrug-resistance (MDR) in bacteria. AcrB is the prototypical member of the RND family and acts to recognise and export a wide range of chemically distinct molecules out of bacteria, conferring resistance to a variety of antibiotics. Although high resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown, preventing a complete mechanistic understanding of efflux and inhibition. Here, we determine these structural dynamics in the presence of AcrB substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular modelling, drug binding and bacterial susceptibility studies. We show that the well-studied efflux pump inhibitor phenylalanine-arginine-{beta}-naphthylamide (PA{beta}N) potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within an MDR clinical isolate, AcrBG288D, modifies the plasticity of the transport pathway, which could explain its altered substrate specificity. Our results provide molecular insight into drug export and inhibition of a major MDR-conferring efflux pump and the important directive role of its dynamics.

biochemistry↗