Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry.

ABSTRACT: Insertion of lipopolysaccharide (LPS) into the outer membrane (OM) of Gram-negative bacteria is mediated by a druggable OM translocon consisting of a beta-barrel membrane protein, LptD, and a lipoprotein, LptE. The beta-barrel assembly machinery (BAM) assembles LptD together with LptE to form a plug-and-barrel structure. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls LPS translocon activation. Here we report the discovery of LptM (formerly YifL), a conserved lipoprotein that assembles together with LptD and LptE at the BAM complex. We demonstrate that LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds and be released as mature LPS translocon by the BAM complex. Inactivation of LptM causes the accumulation of non-natively oxidized LptD, making disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that, by mimicking LPS binding, LptM facilitates oxidative maturation of LptD, thereby activating the LPS translocon.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Escherichia Coli

SUBMITTER: Julien Marcoux

LAB HEAD: Julien Marcoux

PROVIDER: PXD041774 | Pride | 2023-10-11

REPOSITORIES: pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	LptDE-vs-LptDEM-cluster.csv	Csv
	LptDE-vs-LptDEM-cluster_Max_Deuteration.csv	Csv
	LptDE-vs-LptDEM-state.csv	Csv
	LptDEM.fasta	Fasta
	LptDEM_PLGS_final_peptide.zip	Other

Items per page:

1 - 5 of 50

Similar Datasets

Dynamics of an LPS translocon induced by substrate and an antimicrobial peptide

Project description:We investigated the conformational dynamics of the LPS translocon LptDE was using hydrogen-deuterium exchange mass spectrometry. We evaluated the conformational changes of LptDE upon binding of LPS, Re-LPS (an LPS substructure), thanatin, and the membrane lipid POPG. Our data reveal that LPS induces opening of the LptD β-taco domain, coupled with conformational changes on β-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilises the β-taco, thus blocking these conformational fluctuations and preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both β-barrel and β-taco domains.

2020-11-16 | PXD021743 | Pride

Evidence for a widespread third system for bacterial polysaccharide export across the outer membrane comprising a composite OPX/β-barrel translocon

Project description:Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein WzaE. coli is an octamer, in which the eight C-terminal domains form an α-helical OM pore, and the eight copies of the three N-terminal domains (D1-D3) a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18-stranded β-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments and computational structural biology, we characterize EpsX as an OM 18-stranded β-barrel protein important for EPS synthesis and identify AlgE, a β-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, only consists of the periplasmic D1 and D2 domains and lacks the domain for spanning the OM (henceforth D1D2OPX protein). In vivo, EpsX and EpsY mutually stabilize each other, supporting their direct interaction. Based on these observations, we propose a model whereby EpsY and EpsX make up a novel type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning translocon together with the periplasmic D1D2OPX protein EpsY. Based on computational genomics, similar composite systems are present widespread in Gram-negative bacteria. This model provides a framework for these proteins' future experimental characterization.

2023-04-27 | PXD033157 | Pride

Evidence for a widespread third system for bacterial polysaccharide export across the outer membrane comprising a composite OPX/β-barrel translocon

2022-07-12 | PXD035138 | Pride

Understanding the interaction of SurA and BAM in the outer membrane of E. coli

Project description:The outer membrane (OM) is a formidable barrier that protects Gram-negative bacteria against environmental threats. The correct folding and insertion of outer membrane proteins (OMPs) into the OM requires the essential OM-embedded β-barrel assembly machinery (BAM), a heptameric protein complex in E. coli. OMPs are delivered to BAM by the periplasmic chaperone SurA. However, how the activity of SurA and BAM are coordinated to ensure successful OMP delivery to BAM for folding into the OM remained unclear. We have trapped SurA in the act of OMP delivery to BAM and, via cryoEM, solved the structures of this assembly. By mutating key interaction sites we validate this interaction and use proteomics to determine how this perturbs the proteome of E. coli.

2024-10-01 | PXD046606 | Pride

The role of the periplasmic chaperones SurA, Skp and DegP for fitness, outer membrane integrity, antibiotic susceptibility and virulence of Acinetobacter baumannii AB5075: same-same, but different?

Project description:The outer membrane (OM) of Gram-negative bacteria efficiently protects from harmful environmental stresses such as antibiotics, disinfectants or dryness. Main constituents of the OM are integral OM β-barrel proteins (OMPs). In Gram-negative bacteria such as Escherichia coli (Ec), Yersinia enterocolitica (Ye) and Pseudomonas aeruginosa (Pa), the insertion of OMPs depends on a sophisticated biogenesis pathway. This comprises the SecYEG translocon, which enables inner membrane (IM) passage, the chaperones SurA, Skp and DegP, which facilitate the passage of β-barrel OMPs through the periplasm, and the β-barrel assembly machinery (BAM) which facilitates insertion into the OM. In Ec, Ye and Pa the deletion of SurA is particularly detrimental and leads to a loss of OM integrity, sensitization to antibiotics, and hypovirulence. In search of targets that could be exploited to develop compounds that interfere with OM integrity in Acinetobacter baumannii (Ab), we employed the multidrug-resistant strain AB5075 to generate single gene knockout strains lacking individual periplasmic chaperones. In contrast to Ec, Ye and Pa, AB5075 tolerates the lack of SurA, Skp or DegP with only weak mutant phenotypes. While the double knockout strains surAskp and surAdegP are conditionally lethal in Ec, all double deletions were well tolerated by AB5075. Strikingly, even a triple knockout strain of AB5075, lacking surA, skp and degP, was viable. Our findings underline the extreme adaptibility of Ab and suggest the existence of mechanisms bypassing or acting redundantly to the known OMP biogenesis pathway comprising SurA, Skp and DegP.

2022-08-29 | PXD022004 | Pride

The gain-of-function allele bamAE470K bypasses the essential requirement for BamD in β-barrel outer membrane protein assembly

Project description:The outer membrane (OM) of Gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral -barrel outer membrane proteins (OMPs) are assembled into the OM by the beta-barrel assembly machine (BAM), which is composed of one OMP, BamA, and four lipoproteins, BamBCDE. BamBCE can be removed individually with only minor effects on barrier function. However, depletion of BamA or BamD, the only BAM components conserved in all diderm bacteria, causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamAE470K, that bypasses the requirement for BamD. Although bamD::kan bamAE470K cells exhibit growth defects, they assemble OMPs with surprising robustness. Our results demonstrate that the reactions required for beta-barrel folding and membrane integration are catalyzed solely by BamA.

2020-07-03 | PXD017962 | Pride

Tension mediated mechanical activation and pocket delipidation lead to an analogous MscL channel state

Project description:We present HDX-MS data of WT and L89W TbMscL to interrogate the mechanism of molecular activation in this mechanosensitive channel.

2022-06-07 | PXD021983 | Pride

Role of proteins belonging to the OMP biogenesis machinery for virulence of Yersinia enterocolitica

Project description:The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier protecting Gram-negative species from harmful environmental influences. At the same time it facilitates the import of nutrients, the export of proteins, the passage of signaling molecules and also harbors proteins associated with virulence. Transmembrane traffic particularly is facilitated by membrane-integral proteins. In order to insert these into the OM, an essential oligomeric membrane-associated protein complex, the beta-barrel assembly machinery (BAM) is required. Being essential for the biogenesis of outer membrane proteins (OMPs) the BAM and also periplasmic chaperones (termed the OMP biogenesis machinery as an entity herein) may serve as attractive targets to develop novel antimicrobial or antiinfective agents. We aimed to elucidate which proteins belonging to the OMP biogenesis machinery have the most important function in granting bacterial fitness, facilitating biogenesis of dedicated virulence factors and determination of overall virulence. To this end we used the enteropathogen Yersinia enterocolitica (Ye) as a model system. We individually knocked out all non-essential components of the BAM (BamB, C and E) as well as the periplasmic chaperones DegP, SurA and Skp. In summary, we found that the most profound phenotypes were produced by the loss of BamB or SurA with both knockouts resulting in significant attenuation or even avirulence of Ye in a mouse infection model. Thus, both BamB and SurA are promising targets for the development of new antimicrobials in the future.

2018-10-17 | PXD004429 | Pride

Hydrogen-deuterium exchange mass spectrometry analysis of the CDK2-cyclin A-CDC25A complex

Project description:The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of cyclin-dependent protein kinases (CDKs) to activate CDK activity. Here, we present HDX-MS data to complement the cryogenic-electron microscopy (cryo-EM) structure of CDK2-cyclin A in complex with CDC25A, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. This work improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.

2024-05-29 | PXD050866 | Pride

Bacillus subtilis RNase M5 cleaves dsRNA in two steps via a two Mg2+-ion dependent mechanism

Project description:Here, we present the crystal structure of the catalytic domain of M5 bound to two magnesium ions, which reveals the role of each catalytic site acidic residue in metal binding. We further use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the possibility of structural rearrangements that would allow cleavage of the 5’-strand, and use small-angle X-ray scattering (SAXS) to study the M5 binding to the pre-5S rRNA substrate. This study provides new details on the M5 mechanism used for cleavage of the dsRNA substrate, which proceeds via two metal ions and structural rearrangements of both M5 and the pre-5S rRNA substrate.

2021-02-10 | PXD019202 | Pride