Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation

Botte, Mathieu; Ni, D.R.; Schenck, Stephan; Zimmermann, Iwan; Chami, Mohamed; Bocquet, Nicolas; Egloff, Pascal; Bucher, Denis; Trabuco, Matilde Cardoso; Cheng, R.K.; Brunner, Janine D.; Seeger, Markus A.; Stahlberg, Henning; Hennig, Michael

doi:10.1038/s41467-022-29459-2

Cited by 21 publications

(23 citation statements)

References 50 publications

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“…DtpC in complex with Nb17 and Nb26 yielded crystals in various conditions, but despite extensive optimization efforts, the crystals of the DtpC-Nb26 complex did not diffract X-rays better than 5 Å resolution. In a second step, we decided to increase the size of Nb26, which formed a tight complex with DtpC, by fusing one copy of the maltose binding protein (MBP) to its C-terminus as described previously ( Botte et al, 2022 ) . This resulted in a 52 kDa Pro-macrobody (short Mb26), and we expected it to bind to the periplasmic side of the transporter as seen in other MFS transporter-Nb complexes ( Figure 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Considering that the transporter displays no characteristic cytoplasmic or periplasmic features which are helpful to drive the particle alignment, we applied different strategies previously described in the literature to increase the overall size of the transporter to overcome these limitations. We i) fused the transporter to split-sfGFP ( Liu et al, 2020 ; Liu et al, 2022 ), ii) raised different nanobodies against DtpC ( Pardon et al, 2014 ) and iii) extended the nanobody to a Pro-macrobody ( Brunner et al, 2020 ; Botte et al, 2022 ). The various samples were subsequently imaged by cryo-EM and analysed.…”

Section: Introductionmentioning

confidence: 99%

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Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C

Killer

Finocchio

Mertens

et al. 2022

Front. Mol. Biosci.

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Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The E. coli genome codes for four different POTs, known as Di- and tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa Pro-macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 Å. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C

Killer

Finocchio

Mertens

et al. 2022

Front. Mol. Biosci.

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“…We therefore sought to obtain an atomic structure of pa LptDE-YifL and uncover the interaction details. Pa LptD bears a low sequence identity (<25%) with the two LptD homologues with full-length structures to date 21,22,33 , containing an extra 120-residue loop at the N-terminus (Fig. 2A and Extended Data Fig.…”

Section: Resultsmentioning

confidence: 99%

Lipoprotein sorting to the cell surface via a crosstalk between the Lpt and Lol pathways during outer membrane biogenesis

Luo

Wang

Qiao

et al. 2022

Preprint

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Lipopolysaccharide (LPS) and lipoprotein, two essential components of the outer membrane (OM) in Gram-negative bacteria, play critical roles in bacterial physiology and pathogenicity. LPS translocation to the OM is mediated by LptDE, yet how lipoproteins sort to the cell surface remains elusive. Here we report the identification of an inventory of lipoproteins that are transported to the cell surface via LptDE. Notably, we determined crystal structures of LptDE from Pseudomonas aeruginosa and its complex with an endogenous Escherichia coli lipoprotein YifL. The paLptDE-YifL structure demonstrates that YifL translocates to the OM via LptDE, in a manner similar to LPS transport. The β-barrel domain serves as a passage for the proteinaceous moiety while its acyl chains are transported outside. Our finding has been corroborated by results from native mass spectrometry, immunofluorescence, and photocrosslinking assays, revealing a unique mechanism through which lipoproteins are translocated across the OM in an ATP- and LPS-dependent manner. Moreover, our study expands the scope of current knowledge of lipoprotein sorting by disclosing a crosstalk between the Lpt and Lol pathways.

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“…Given that LPS is integrated unipolarly in the OM (Vassen et al, 2019), we wonder whether the LPS translocation pathway was also localized at the growing cell pole. Together with LptE, LptD forms the OM part of the Lpt pathway, allowing the last step of the LPS transport to the OM (Wu et al, 2006;Botte et al, 2022).In order to localize this large beta barrel in B. abortus, a 3Flag tag was incorporated into an unconserved loop of LptD (Fig. S1), predicted to be surface exposed (Fig.…”

Section: The Om Component Of the Lpt Pathway Lptd Is Dispersed On B A...mentioning

confidence: 99%

Lipopolysaccharide synthesis and traffic in the envelope of the pathogen Brucella abortus

Servais

Vassen

Verhaeghe

et al. 2022

Preprint

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Lipopolysaccharide is essential for most Gram-negative bacteria as it is a main component of the outer membrane. In the pathogen Brucella abortus, smooth lipopolysaccharide containing the O-antigen is required for virulence. Being part of the Rhizobiales, Brucella spp. display unipolar growth and lipopolysaccharide was shown to be incorporated at the active growth sites, i.e. the new pole and the division site. By localizing proteins involved in the lipopolysaccharide transport across the cell envelope, from the inner to the outer membrane, we show that the lipopolysaccharide incorporation sites are determined by the inner membrane complex of the lipopolysaccharide transport system. Moreover, we identify the main O-antigen ligase of Brucella spp involved in smooth lipopolysaccharide synthesis. Altogether, our data highlight a new layer of spatiotemporal organization of the lipopolysaccharide biosynthesis pathway and identify a new class of bifunctional O-antigen ligases.

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Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation

Cited by 21 publications

References 50 publications

Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C

Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C

Lipoprotein sorting to the cell surface via a crosstalk between the Lpt and Lol pathways during outer membrane biogenesis

Lipopolysaccharide synthesis and traffic in the envelope of the pathogen Brucella abortus

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