Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are major drivers of multidrug resistance among Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, it’s pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the structural dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug 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 instead in domain specific organisation. We establish a unique histidine residue directs these dynamics and is essential for sustaining pump activity across acidic, neutral, and basic regimes. Overall, we propose Mg2+ conserves AcrA structural mobility to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced during bacterial infection and colonization.

INSTRUMENT(S): Xevo G2 Q-Tof, Synapt MS

ORGANISM(S): Escherichia Coli

SUBMITTER: Benjamin Russell Lewis

LAB HEAD: Eamonn Reading

PROVIDER: PXD057200 | Pride | 2024-12-30

REPOSITORIES: pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	210217_ls_i_HDX_AcrA_NSP_pH6_0sec_01_IA_final_peptide.csv	Csv
	210217_ls_i_HDX_AcrA_NSP_pH6_0sec_02_IA_final_peptide.csv	Csv
	210217_ls_i_HDX_AcrA_NSP_pH6_0sec_03_IA_final_peptide.csv	Csv
	210217_ls_i_HDX_AcrA_NSP_pH6_0sec_04_IA_final_peptide.csv	Csv
	210412_ls_i_HDX_AcrA_NSP_ph74_0sec_01_IA_final_peptide.csv	Csv

Items per page:

1 - 5 of 35

Similar Datasets

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

Project description: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 αβ 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.

2023-06-01 | PXD041359 | Pride

Allosteric activation of the co-receptor BAK1 by the EFR receptor kinase initiates immune signaling

Project description:The structural dynamics of purified wild type and mutant EFR were probed by HDX-MS.

2024-06-27 | PXD049215 | Pride

Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation

Project description:Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a complex, multi-stage activation process. Ca2+-flux induces conformational rearrangements that relieve autoinhibitory FERM domain interactions. The kinase domain phosphorylates a key linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loop residues to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. Here, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map the conformational changes associated with Src-mediated activation segment phosphorylation in a Pyk2 construct encompassing FERM and kinase domains (residues 20-692). Results reveal increased dynamics at regulatory interfaces spanning FERM and kinase domains. Phosphorylation of the activation segment stabilizes two antiparallel beta strands linking activation and catalytic loops. Increased dynamics of the C-terminal end of the activation loop propagate to the F-helix, explaining how phosphorylation prevents reversion to the autoinhibitory FERM interaction. HDX-guided site-directed mutagenesis and kinase activity profiling establish a mechanism for phosphorylation-induced active site sculpting to confer high activity.

2023-03-08 | PXD034028 | Pride

The strutural dynamics of EGFR Exon 19 mutants

Project description:Tyrosine kinase inhibtors are used to treat non-small cell lung cancers (NSCLC) driven by epidermal growth factor receptor (EGFR) mutations in the tyrosine kinase domain (TKD). We characterized the dynamics of purified EGFR exon 19 kinase domain mutants by HDX-MS with or withour erlotinib, one of the first-generation TKIs.

2023-03-11 | PXD037448 | Pride

HDX-MS of Activators and Inhibitors of PI3Kalpha

Project description:Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI 3-kinase (PI3K) signalling pathway which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation1-3. Here we report on the discovery of UCL-TRO-1938 (further referred to as 1938), a small molecule activator of the PI3Kα isoform, a critical effector of growth factor signalling. 1938 allosterically activates PI3Kα through a unique mechanism, by enhancing multiple steps of the PI3Kα catalytic cycle, and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Ka over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia reperfusion injury and, upon local administration, enhances nerve regeneration following nerve crush. This study identifies a unique chemical tool to directly probe PI3Kα signalling and a novel approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes, through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

2023-03-06 | PXD037721 | Pride

Conformational plasticity of RAS Q61 family of neoepitopes results in distinct features for targeted recognition.

Project description:We utilize hydrogen deuterium exchange mass spectrometry (HDX-MS) to probe the solvation and conformational dynamics of HLA-A*01:01/hβ2m complexes that are emptied or loaded with NRAS Q61K peptide, as well as wild-type (Q61) and three additional, common neoepitopes (Q61H, Q61L, Q61R).

2023-11-30 | PXD044838 | Pride

HDX-MS on secondary transporters

Project description:Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane – yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters.

2018-10-16 | PXD011060 | Pride

Selective modulation of nuclear receptor LRH-1 through target-ing buried and surface regions of the binding pocket

Project description:Liver receptor homolog-1 (LRH-1) is a phospholipid-sensing nuclear receptor that has shown promise as a target for alleviat-ing intestinal inflammation and disease states characterized by metabolic dysregulation in the liver. LRH-1 contains an unu-sually large ligand binding pocket, and generating synthetic modulators has been challenging. Leveraging a hexahydropen-talene (6HP) core, we have had recent success in generating potent and efficacious agonists through two distinct strategies. We targeted residues deep within the pocket to enhance compound binding, while residues at the mouth of the pocket were targeted to mimic interactions made by endogenous phospholipids. Here, we unite these two designs into one hybrid mole-cule that is the most potent LRH-1 agonist to date. Through a combination of global transcriptomic, biochemical, and struc-tural studies, we show that selective modulation can be driven through contacting deep vs. surface polar regions in the pock-et. While deep pocket contacts convey high-affinity, contacts with the pocket mouth dominate allostery and provide a phos-pholipid-like transcriptional response in cultured cells.

2022-05-04 | PXD031099 | Pride

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro (HDX-MS pulse labeling)

Project description:SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic, entailing enormous disruption worldwide. Its ~30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein, and the data suggest that the nsp7-10/11 structure in complex with Mpro strongly resembles the unbound polyprotein. Our array of techniques supports the notion that interplay between polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we leveraged assays of limited proteolysis by Mpro of nsp7-11 using a series of inhibitors/binders to characterize Mpro inhibition using a polyprotein substrate.

2022-12-19 | PXD033702 | Pride

Patient-derived anti-Siglec-6 antibodies engineered into T cell-recruiting bispecific antibody formats for treating leukemia

Project description:Despite numerous therapeutic options, safe and curative therapy is out of reach for most chronic lymphocytic leukemia (CLL) patients. We previously reported the identification of a patient-derived CLL-binding antibody, JML-1, and identified Siglec-6 as the target of JML-1. Siglec-6 is an attractive target for cancer immunotherapy due to its absence on healthy tissues and potential role in immune signaling. In this work, we identify additional patient-derived anti-Siglec-6 antibodies, RC-1 and RC-2, that bind with higher affinity than JML-1, yet exhibit similar specificity and overlapping conformational epitopes. Both JML-1 and RC-1 were employed to generate anti-Siglec-6 x anti-CD3 T cell-recruiting bispecific antibodies (T-biAbs) which were effective in vitro at activating T cells and lysing target cells. Upon engineering RC-1 into more compact and rigid T-biAb formats, we achieved sub-pM EC50 values for cell lysis in vitro. In addition, the T-biAbs mediated the killing of primary CLL cells by autologous T cells at natural effector-to-target cell ratios ex vivo. The increased potency of this T-biAb format appears to be due to the reduction in the length and/or flexibility of the cytolytic synapse. Furthermore, the anti-Siglec-6 x anti-CD3 T-biAb was effective at curing mice in a systemic in vivo model of CLL.

2022-12-19 | PXD029601 | Pride