A lipoprotein partner for the Escherichia coli outer membrane protein TolC
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eLife Assessment
In this fundamental work Horne et al present compelling evidence that YbjP is a novel binding partner of the TolC channel protein. The YbjP is characterized using cryo-EM, and its role probed using pull-down experiments, in vivo crosslinking, functional assays along with phylogenetic analysis which are all properly performed and presented and support the main conclusions. While the study does not identify a clear role for this protein, the results contribute to the understanding of this complex system and will be of interest to those working in the fields of membrane transport and antimicrobial resistance.
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Abstract
The outer-membrane protein TolC from Escherichia coli belongs to an extensive superfamily whose members are found throughout the didermal, Gram-negative bacterial lineages. The protein serves as an activated exit duct in multi-drug efflux pumps and protein secretion machinery. Many TolC homologs bear a lipid modification on the N-terminus that embeds into the inner leaflet of the outer membrane and appears to have been a conserved feature for millions of years; however, the moiety is absent entirely in the E. coli TolC. We have discovered that the E. coli lipoprotein YbjP interacts extensively with the periplasmic surface of TolC and its N-terminal lipid moiety is embedded in the membrane, mimicking the intramolecular interactions seen for related proteins. Here, we present cryo-EM structures of the MacA-MacB-TolC and AcrA-AcrB-TolC tripartite pumps complexed to YbjP. We demonstrate that the association occurs spontaneously both in vitro and in vivo and that YbjP facilitates recovery following exposure to bacteriostatic agents. We suggest that the YbjP-TolC interaction may facilitate the assembly of the outer membrane protein in a redundant biogenesis pathway.
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eLife Assessment
In this fundamental work Horne et al present compelling evidence that YbjP is a novel binding partner of the TolC channel protein. The YbjP is characterized using cryo-EM, and its role probed using pull-down experiments, in vivo crosslinking, functional assays along with phylogenetic analysis which are all properly performed and presented and support the main conclusions. While the study does not identify a clear role for this protein, the results contribute to the understanding of this complex system and will be of interest to those working in the fields of membrane transport and antimicrobial resistance.
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Reviewer #1 (Public review):
Summary:
The authors report a novel binding partner of the TolC channel protein that forms complexes with the two principal classes of transporter-based tripartite assemblies (both ABC- and RND-transporter based) and appears to modulate their function, while also anchoring TolC into the outer membrane, compensating for the lack of direct lipidation seen in other members of the OMF family.
The newly identified protein, YbjP, is comprehensively characterized from both phylogenetic and structural perspectives. Two independent cryo-EM structures (MacAB-TolC-YbjP and AcrABZ-TolC-YbjP) provide strong structural evidence for its role and are generated using peptidiscs, mimicking the membrane environment. These findings are further supported by pull-down experiments (including state-of-the-art in vivo photo …
Reviewer #1 (Public review):
Summary:
The authors report a novel binding partner of the TolC channel protein that forms complexes with the two principal classes of transporter-based tripartite assemblies (both ABC- and RND-transporter based) and appears to modulate their function, while also anchoring TolC into the outer membrane, compensating for the lack of direct lipidation seen in other members of the OMF family.
The newly identified protein, YbjP, is comprehensively characterized from both phylogenetic and structural perspectives. Two independent cryo-EM structures (MacAB-TolC-YbjP and AcrABZ-TolC-YbjP) provide strong structural evidence for its role and are generated using peptidiscs, mimicking the membrane environment. These findings are further supported by pull-down experiments (including state-of-the-art in vivo photo crosslinking) and functional assays for a well-rounded characterisation of the protein, and a significant amount of modelling and phylogenetic analysis. This work sheds light on the function of the members of the DUF3828-containing protein family, which appear to anchor TolC to the outer membrane and influence the expression of the TnaB and YojI transporters.
Strengths:
The strengths of the manuscript are numerous, and it presents a well-rounded package of structural biology complemented by functional and computational studies.
The full assemblies of both MacAB-TolC-YbjP and AcrABZ-TolC-YbjP are reconstituted and resolved to near-atomic resolution using cryo-EM for unambiguous assignment of binding interfaces, which are then validated using a number of techniques, including ITC, in vitro and in vivo binding assays and cross-linking.
The evolutionary analysis is particularly notable, and provides genuine insight into the DUF3828-containing proteins, the function of which remains enigmatic till now. Similarly, the involvement of YbjP in trafficking of TolC and the analysis of the impact of YbjP deletion of the full E. coli proteome is commendable.
Overall, this is a very solid piece of work, competently executed and presented, which significantly advances the field.
Weaknesses:
None obvious, however the presentation and especially main-text illustrative material seems to focus disproportionately on MacAB-TolC-YbjP complex, and the AcrABZ-TolC-YbjP is relegated to supplementary data which is somewhat confusing. There is no high-resolution side view of the AcrABZ-TolC-YbjP side-by-side to MacAB-TolC-YbjP which may be helpful to spot parallels and differences in the organisation of the two systems.
Supplementary Figure 2 may also be better presented in the main text, as it shows specific displacements of residues upon binding of the YbjP relative to the apo-complexes, although this can be left at the authors' discretion.
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Reviewer #2 (Public review):
This article focuses on the study of two E. coli tripartite efflux pumps both using TolC as partner in the outer membrane, namely MacAB-TolC and AcrABZ-TolC.
By preparing MacAB-TolC in Peptidiscs rather than in detergent for cryo-EM structure determination, they visualized an extra protein localized around TolC. The resolution was sufficient to build part of the structure, and using the AlphaFold2 database and DALI topology recognition program, they identified it as the lipoprotein YbjP. This protein has an anchorage in the outer membrane, and it was suggested that it could act as a support for TolC that is the only OMF that does not have an N-terminal extension anchored in the outer membrane, which is very puzzling for the community working in this field of research.
Authors used a large number of different …
Reviewer #2 (Public review):
This article focuses on the study of two E. coli tripartite efflux pumps both using TolC as partner in the outer membrane, namely MacAB-TolC and AcrABZ-TolC.
By preparing MacAB-TolC in Peptidiscs rather than in detergent for cryo-EM structure determination, they visualized an extra protein localized around TolC. The resolution was sufficient to build part of the structure, and using the AlphaFold2 database and DALI topology recognition program, they identified it as the lipoprotein YbjP. This protein has an anchorage in the outer membrane, and it was suggested that it could act as a support for TolC that is the only OMF that does not have an N-terminal extension anchored in the outer membrane, which is very puzzling for the community working in this field of research.
Authors used a large number of different approaches to evaluate the importance of YbjP (structure, genomic evolution, microbiology, photocrosslink in vivo, proteomic profile), but did not succeed in finding it a clear role so far, even if it could be important depending on environmental stress. Nevertheless, their results are of main interest for the comprehension of the complexity of such systems and deserve publication.
The different analyses are properly performed and presented, and support the conclusions.
My only concern is for the photocrosslink presented in Figures 3 and S3. My impression is that the bands do not migrate at the proper size after the crosslink.
A second point that could be discussed further is the comparison of the structure of the pump in the presence of the peptidoglycan with the images previously obtained by tomography. It is not totally clear to me if YbjP could have been positioned in these maps.
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