Structural insight into the formation of lipoprotein-β-barrel complexes by the β-barrel assembly machinery

Létoquart, Juliette; Rodríguez-Alonso, Raquel; Vs, Nguyen; Louis, Gwennaelle; An, C.; Se, Radford; Cho, Soyun; Remaut, Han; Collet, Jean-François

doi:10.1101/823146

Cited by 4 publications

(2 citation statements)

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“…Recently, the crystal structure of BamA in complex with RcsF was solved, providing insight into the mechanism of surface exposure of RcsF. 22 Considering that this mechanism is BAM-dependent, any perturbation of the BAM complex is expected to induce Rcs stress. Consistently, the Rcs system is induced in a bamA knockdown strain 19 , 23 or by addition of the BAM inhibitor darobactin.…”

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confidence: 99%

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Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

et al. 2021

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The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the β-barrel assembly machine (BAM), located in the OM and responsible for β-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σ E and Rcs stress in Escherichia coli . Screening a library consisting of 316 953 compounds yielded five compounds that induced σ E and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.

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confidence: 99%

“…Recently, the crystal structure of BamA in complex with RcsF was solved, providing insight into the mechanism of surface exposure of RcsF . Considering that this mechanism is BAM-dependent, any perturbation of the BAM complex is expected to induce Rcs stress.…”

mentioning

confidence: 99%

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

et al. 2021

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Systematic localization of Gram-negative bacterial membrane proteins

Sueki¹,

Stein²,

Savitski³

et al. 2019

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14The molecular architecture and function of the Gram-negative bacterial cell envelope 15 is dictated by protein composition and localization. Proteins that localize to the inner 16 (IM) and outer (OM) membranes of Gram-negative bacteria play critical and distinct 17 roles in cellular physiology, however, approaches to systematically interrogate their 18 distribution across both membranes and the soluble cell fraction are lacking. We 19 employed multiplexed quantitative mass spectrometry to assess membrane protein 20 localization in a proteome-wide fashion by separating IM and OM vesicles from 21 exponentially growing E. coli K-12 cells on a sucrose density gradient. The migration 22 54 Proteins localizing to the IM or OM can be sub-categorized based on their distinct 55 biophysical properties. IM proteins typically contain α-helical transmembrane 56 domains that mediate their integration into the phospholipid bilayer. These proteins 57 include small molecule transporters (e.g. ABC transporters for metal ions and sugars) 58 and large multi-protein complexes (e.g. SecYEG translocon and ATP synthase). IM 59 proteins are diverse in their structure, function and domain localization; some contain 60 structural domains that extend into the cytoplasmic and/or periplasmic space. In the 61 OM, there are two distinct types of proteins: outer membrane proteins (OMPs), which 62 are mostly composed of amphipathic β-strands that form a closed β-barrel structure, 63and lipoproteins, which are anchored to the OM via an N-terminal lipidated cysteine 64 and typically contain a soluble domain that extends into the periplasmic space. 65

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Systematic Localization of Escherichia coli Membrane Proteins

et al. 2020

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The molecular architecture and function of the Gram-negative bacterial cell envelope are dictated by protein composition and localization. Proteins that localize to the inner membranes (IM) and outer membranes (OM) of Gram-negative bacteria play critical and distinct roles in cellular physiology; however, approaches to systematically interrogate their distribution across both membranes and the soluble cell fraction are lacking. Here, we employed multiplexed quantitative mass spectrometry using tandem mass tag (TMT) labeling to assess membrane protein localization in a proteome-wide fashion by separating IM and OM vesicles from exponentially growing Escherichia coli K-12 cells on a sucrose density gradient. The migration patterns for Ͼ1,600 proteins were classified in an unbiased manner, accurately recapitulating decades of knowledge in membrane protein localization in E. coli. For 559 proteins that are currently annotated as peripherally associated with the IM (G. Orfanoudaki and A. Economou, Mol Cell Proteomics 13:3674 -3687, 2014, https://doi.org/ 10.1074/mcp.O114.041137) and that display potential for dual localization to either the IM or cytoplasm, we could allocate 110 proteins to the IM and 206 proteins to the soluble cell fraction based on their fractionation patterns. In addition, we uncovered 63 cases, in which our data disagreed with current localization annotation in protein databases. For 42 of these cases, we were able to find supportive evidence for our localization findings in the literature. We anticipate that our systems-level analysis of the E. coli membrane proteome will serve as a useful reference data set to query membrane protein localization, as well as to provide a novel methodology to rapidly and systematically map membrane protein localization in more poorly characterized Gram-negative species. IMPORTANCE Current knowledge of protein localization, particularly outer membrane proteins, is highly dependent on bioinformatic predictions. To date, no systematic experimental studies have directly compared protein localization spanning the inner and outer membranes of E. coli. By combining sucrose density gradient fractionation of inner membrane (IM) and outer membrane (OM) proteins with multiplex quantitative proteomics, we systematically quantified localization patterns for Ͼ1,600 proteins, providing high-confidence localization annotations for 1,368 proteins. Of these proteins, we resolve the predominant localization of 316 proteins that currently have dual annotation (cytoplasmic and IM) in protein databases and identify new annotations for 42 additional proteins. Overall, we present a novel quantitative methodology to systematically map membrane proteins in Gram-negative bacteria and use it to unravel the biological complexity of the membrane proteome architecture in E. coli.

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Structural insight into the formation of lipoprotein-β-barrel complexes by the β-barrel assembly machinery

Cited by 4 publications

References 43 publications

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

Systematic localization of Gram-negative bacterial membrane proteins

Systematic Localization of Escherichia coli Membrane Proteins

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