Structure of an endogenous mycobacterial MCE lipid transporter

Chen, James; Fruhauf, Alice; Fan, Catherine; Ponce, Jackeline; Ueberheide, Beatrix; Bhabha, Gira; Ekiert, D.C.

doi:10.1101/2022.12.08.519548

Cited by 4 publications

(4 citation statements)

References 94 publications

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“…We suspect that the "open groove" configuration may represent an artifact of the prediction, owing to the way AlphaFold-Multimer concatenates sequences of all chains prior to structure inference. Instead, the closed tunnel model (Extended Fig 5) more closely reflects the resolved crystal structure of PqiB, another MCE-domain protein from E. coli, which forms a closed α-helical tunnel on top of three hexameric MCE rings 6 , and the recently resolved heterohexamer of Mce1A-1F from Mycobacterium tuberculosis 30 . Like these resolved structures, the predicted tunnel formed by the α-helices generates a hydrophobic interior of ~13 Å (Fig 4C ), supporting the possibility of a role in hydrophobic substrate transport.…”

Section: Fig 4)mentioning

confidence: 79%

“…tuberculosis 30 have been shown to form both hetero-and homohexamers, and for two of these structures (PqiB and Mce1A-1F), the hexameric nature of the α-helical region results in a closed tunnel that spans the periplasm, with a hydrophobic interior. These studies suggest the tunnel structures could facilitate the transport of a hydrophobic substrate and, from homology, conservation and phenotypic characterisation, the proposed substrate is a glycerophospholipid (or in the case of M. tuberculosis, cholesterol).…”

Section: Discussionmentioning

confidence: 99%

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Bridges instead of boats? The Mla system of diderm FirmicuteVeillonella parvulareveals an ancestral transenvelope core of phospholipid trafficking

Grasekamp,

Beaud,

Taib

et al. 2023

Preprint

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Despite extensive characterisation of envelope biogenesis systems in diderm bacteria, glycerophospholipid (GPL) trafficking remains poorly understood, and has only been studied in a handful of model species. Within the Proteobacteria, the maintenance of lipid asymmetry (Mla) system facilitates retrograde GPL trafficking via six proteins, MlaA-F. GPLs are extracted from the outer leaflet of the outer membrane by the lipoprotein MlaA which associates with porin trimers, then shipped through the periplasmic space by the chaperone MlaC, which finally delivers GPLs to the inner membrane complex formed by MlaBDEF. Here, we investigate GPL trafficking in Veillonella parvula, a diderm member of the Firmicutes which encodes an Mla system devoid of MlaA and MlaC. V. parvula mla mutants display phenotypes characteristic of disrupted lipid asymmetry such as hypervesiculation and detergent hypersensitivity, and lipid content analysis from outer membrane vesicles reveals an enrichment for the major lipid component phosphatidylethanolamine. Interestingly, suppressor analysis identifies mutations in tamB that rescue detergent hypersensitivity and hypervesiculation of delta mla strains, supporting the involvement of these two systems in antagonistic GPL trafficking functions across diverse bacterial lineages. A combination of structural modeling and subcellular localisation assays shows that MlaDVp is longer than in classical diderm models and forms a transenvelope bridge, encoding both an inner membrane-localised MCE domain and an outer membrane beta-barrel. These results strongly suggest that V. parvula possesses a minimal Mla system for GPL trafficking, replacing the need for chaperones and outer membrane lipoproteins by directly connecting the two membranes. Finally, phylogenomic analysis indicates that this MlaEFD self-contained architecture is widely distributed in diderm bacteria and most likely represents the ancestral functional core of the Mla system, which subsequently increased in complexity in Proteobacteria and closely related phyla following the emergence of MlaABC. Our work broadens the diversity of current models of GPL trafficking in diderm bacteria, challenging the paradigm set by classical models and shedding light on the evolution of a crucial system in the biogenesis and maintenance of the bacterial outer membrane.

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Section: Fig 4)mentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Bridges instead of boats? The Mla system of diderm FirmicuteVeillonella parvulareveals an ancestral transenvelope core of phospholipid trafficking

Grasekamp,

Beaud,

Taib

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Recently, a cryo-EM structure of the Mycobacterium smegmatis Mce1 transporter was reported. Our proteomic approach identified homologs for the each of the core Mce1 proteins in Mtb including Rv2536/LucB ( 18 ). Importantly, Rv2536/LucB was previously not associated with the Mce1 transporter.…”

Section: Resultsmentioning

confidence: 99%

MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis

Fieweger

Wilburn

Montague

et al. 2023

Journal of Biological Chemistry

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“…One particularly powerful application of this protocol is generating samples for structural analysis, which requires large scale production of highly pure samples 2, 7 . For example, we have used this strategy to purify the nine-subunit human ER membrane protein complex (EMC), for structure determination using single particle cryo-EM 2 .…”

Section: Introductionmentioning

confidence: 99%

A nanobody-based strategy for rapid and scalable purification of native human protein complexes

Stevens

Tomaleri

Hazu

et al. 2023

Preprint

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Native isolation of proteins in high yield and purity is a major bottleneck for analysis of their three-dimensional structure, function, and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly-specific nanobody-mediated purification and proteolytic elution. (1) First, generation of a plasmid coding for a protein of interest fused to an N- or C-terminal GFP or ALFA peptide tag is rapidly achieved using the lentiviral plasmid toolkit we have designed. (2) Human suspension cell lines stably expressing the tagged fusion protein can be generated in <5 days using lentiviral transduction. (3) Leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, proteins expressed even at low levels can be specifically captured from the resulting cell lysate in a variety of conditions, including detergents and mild denaturants. (4) Finally, rapid and specific elution of tagged or untagged proteins under native conditions is achieved within minutes at 4˚C using an engineered SUMO protease. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP expression plasmids, cell lines and transgenic model organisms; is faster than alternative approaches, requiring ~8 days from cloning to purification; and results in substantially improved yields and purity.

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Structure of an endogenous mycobacterial MCE lipid transporter

Cited by 4 publications

References 94 publications

Bridges instead of boats? The Mla system of diderm FirmicuteVeillonella parvulareveals an ancestral transenvelope core of phospholipid trafficking

Bridges instead of boats? The Mla system of diderm FirmicuteVeillonella parvulareveals an ancestral transenvelope core of phospholipid trafficking

MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis

A nanobody-based strategy for rapid and scalable purification of native human protein complexes

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