Structural mechanisms of pump assembly and drug transport in the AcrAB-TolC efflux system

Reviewer #1 (Public review):

Summary:

This manuscript investigates the biological mechanism underlying the assembly and transport of the AcrAB-TolC efflux pump complex. By combining endogenous protein purification with cryo-EM analysis, the authors show that the AcrB trimer adopts three distinct conformations simultaneously and identify a previously uncharacterized lipoprotein, YbjP, as a potential additional component of the complex. The work aims to advance our understanding of the AcrAB-TolC efflux system in near-native conditions and may have broader implications for elucidating its physiological mechanism.

Strengths:

Overall, the manuscript is clearly presented, and several of the datasets are of high quality. The use of natively isolated complexes is a major strength, as it minimizes artifacts associated with reconstituted systems and enables the discovery of a novel subunit. The authors also distinguish two major assemblies-the TolC-YbjP sub-complex and the complete pump-which appear to correspond to the closed and open channel states, respectively. The conceptual advance is potentially meaningful, and the findings could be of broad interest to the field.

Weaknesses:

(1) As the identification of YbjP is a key contribution of this work, a deeper comparison with functional "anchor" proteins in other efflux pumps is needed. Including an additional supplementary figure illustrating these structural comparisons would be valuable.

(2) The observation of the LTO states in the presence of TolC represents an important extension of previous findings. A more detailed discussion comparing these LTO states to those reported in earlier structural and biochemical studies would improve the clarity and significance of this point.

Reviewer #2 (Public review):

Summary:

This manuscript reports the high-resolution cryo-EM structures of the endogenous TolC-YbjP-AcrABZ complex and a TolC-YbjP subcomplex from E. coli, identifying a novel accessory subunit. This work is an impressive effort that provides valuable structural insights into this native complex.

Strengths:

(1) The study successfully determines the structure of the complete, endogenously purified complex, marking a significant achievement.

(2) The identification of a previously unknown accessory subunit is an important finding.

(3) The use of cryo-EM to resolve the complex, including potential post-translational modifications such as N-palmitoyl and S-diacylglycerol, is a notable highlight.

Weaknesses:

(1) Clarity and Interpretation: Several points need clarification. Additionally, the description of the sample preparation method, which is a key strength, is currently misplaced and should be introduced earlier.

(2) Data Presentation: The manuscript would benefit significantly from improved figures.

(3) Supporting Evidence: The inclusion of the protein purification profile as a supplementary figure is essential. Furthermore, a discussion comparing the endogenous AcrB structure to those obtained in other systems (e.g., liposomes) and commenting on observed lipid densities would strengthen the overall analysis.

Reviewer #3 (Public review):

Summary:

The manuscript "Structural mechanisms of pump assembly and drug transport in the AcrAB-TolC efflux system" by Ge et al. describes the identification of a previously uncharacterized lipoprotein, YbjP, as a novel partner of the well-studied Enterobacterial tripartite efflux pump AcrAB-TolC. The authors present cryo-electron microscopy structures of the TolC-YbjP subcomplex and the complete AcrABZ-TolC-YbjP assembly. While the identification and structural characterization of YbjP are potentially novel, the stated focus of the manuscript-mechanisms of pump assembly and drug transport - is not sufficiently addressed. The manuscript requires reframing to emphasize the principal novelty associated with YbjP and significant development of the other aspects, especially the claimed novelty of the AcrB drug-efflux cycle.

Strengths:

The reported association of YbjP with AcrAB-TolC is novel; however, a recent deposition of a preceding and much more detailed manuscript to the BioRxiv server (Horne et al., https://doi.org/10.1101/2025.03.19.644130) removes much of the immediate novelty.

Weaknesses:

While the identification of YbjP is novel, the authors do not appear to acknowledge the precedence of another work (Horne et al., 2025), and it is not cited within the correct context in the manuscript.

Several results presented in the TolC-YbjP section do not represent new findings regarding TolC structure itself. The structure and gating behaviour of TolC should be more thoroughly introduced in the Introduction, including prior work describing channel opening and conformational transitions. The current manuscript does not discuss the mechanistic role of helices H3/H4 and H7/H8 in channel dilation, despite implying that YbjP binding may influence these features. Only the original closed TolC structure is cited, and the manuscript does not address prior mutational studies involving the D396 region, though this residue is specifically highlighted in the presented structures.

The manuscript provides only a general structural alignment between the closed TolC-YbjP subcomplex and the open TolC observed in the full pump assembly. However, multiple open, closed, and intermediate conformations of AcrAB-TolC have already been reported. Thus, YbjP alone cannot be assumed to account for TolC channel gating. A systematic comparison with existing structures is necessary to determine whether YbjP contributes any distinct allosteric modulation.

The analysis of AcrB peristaltic action is superficial, poorly substantiated and importantly, not novel. Several references to the ATP-synthase cycle have been provided, but this has been widely established already some 20 years ago - e.g. https://www.science.org/doi/10.1126/science.1131542.

The most significant limitation of the study is the absence of functional characterization of YbjP in vivo or in vitro. While the structural association between YbjP and TolC is interesting, the biological role of YbjP remains unclear. Moreover, the manuscript does not examine structural differences between the presented complex and previously solved AcrAB-TolC or MexAB-OprM assemblies that might support a mechanistic model.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript investigates the biological mechanism underlying the assembly and transport of the AcrAB-TolC efflux pump complex. By combining endogenous protein purification with cryo-EM analysis, the authors show that the AcrB trimer adopts three distinct conformations simultaneously and identify a previously uncharacterized lipoprotein, YbjP, as a potential additional component of the complex. The work aims to advance our understanding of the AcrAB-TolC efflux system in near-native conditions and may have broader implications for elucidating its physiological mechanism.

Strengths:

Overall, the manuscript is clearly presented, and several of the datasets are of high quality. The use of natively isolated complexes is a major strength, as it minimizes artifacts associated with reconstituted systems and enables the discovery of a novel subunit. The authors also distinguish two major assemblies-the TolC-YbjP sub-complex and the complete pump-which appear to correspond to the closed and open channel states, respectively. The conceptual advance is potentially meaningful, and the findings could be of broad interest to the field.

Weaknesses:

(1) As the identification of YbjP is a key contribution of this work, a deeper comparison with functional "anchor" proteins in other efflux pumps is needed. Including an additional supplementary figure illustrating these structural comparisons would be valuable.

We appreciate this helpful suggestion. We will expand the comparative analysis between YbjP and established anchoring or accessory components in other efflux pumps, and we will add a new supplementary figure illustrating these structural relationships.

(2) The observation of the LTO states in the presence of TolC represents an important extension of previous findings. A more detailed discussion comparing these LTO states to those reported in earlier structural and biochemical studies would improve the clarity and significance of this point.

We agree. In the revised manuscript we will expand our discussion of the LTO conformations, including a direct comparison with previously reported structural and biochemical observations, to better contextualize the significance of our findings.

Reviewer #2 (Public review):

Summary:

This manuscript reports the high-resolution cryo-EM structures of the endogenous TolC-YbjP-AcrABZ complex and a TolC-YbjP subcomplex from E. coli, identifying a novel accessory subunit. This work is an impressive effort that provides valuable structural insights into this native complex.

Strengths:

(1) The study successfully determines the structure of the complete, endogenously purified complex, marking a significant achievement.

(2) The identification of a previously unknown accessory subunit is an important finding.

(3) The use of cryo-EM to resolve the complex, including potential post-translational modifications such as N-palmitoyl and S-diacylglycerol, is a notable highlight.

Weaknesses:

(1) Clarity and Interpretation: Several points need clarification. Additionally, the description of the sample preparation method, which is a key strength, is currently misplaced and should be introduced earlier.

Thank you for pointing this out. We will reorganize the text to introduce the sample preparation strategy earlier and clarify the points that may cause ambiguity.

(2) Data Presentation: The manuscript would benefit significantly from improved figures.

We agree and will revise the figures to improve clarity, consistency, and readability. Additional schematic illustrations will also be included where appropriate.

(3) Supporting Evidence: The inclusion of the protein purification profile as a supplementary figure is essential. Furthermore, a discussion comparing the endogenous AcrB structure to those obtained in other systems (e.g., liposomes) and commenting on observed lipid densities would strengthen the overall analysis.

We appreciate these suggestions. We will add the purification profile and expand the comparison between our endogenous AcrB structure and previously reported structures from reconstituted systems, including a more detailed discussion of lipid densities.

Reviewer #3 (Public review):

Summary:

The manuscript "Structural mechanisms of pump assembly and drug transport in the AcrAB-TolC efflux system" by Ge et al. describes the identification of a previously uncharacterized lipoprotein, YbjP, as a novel partner of the well-studied Enterobacterial tripartite efflux pump AcrAB-TolC. The authors present cryo-electron microscopy structures of the TolC-YbjP subcomplex and the complete AcrABZ-TolC-YbjP assembly. While the identification and structural characterization of YbjP are potentially novel, the stated focus of the manuscript-mechanisms of pump assembly and drug transport - is not sufficiently addressed. The manuscript requires reframing to emphasize the principal novelty associated with YbjP and significant development of the other aspects, especially the claimed novelty of the AcrB drug-efflux cycle.

Strengths:

The reported association of YbjP with AcrAB-TolC is novel; however, a recent deposition of a preceding and much more detailed manuscript to the BioRxiv server (Horne et al., https://doi.org/10.1101/2025.03.19.644130) removes much of the immediate novelty.

Weaknesses:

While the identification of YbjP is novel, the authors do not appear to acknowledge the precedence of another work (Horne et al., 2025), and it is not cited within the correct context in the manuscript.

We thank the reviewer for rasising this important point regarding the independent nature of our work.

Our study indeed progressed independently. The process began with our purification of an endogenous protein sample containing the AcrAB-TolC efflux pump. During our cryo-EM analysis, we observed an unassigned density in the map, for which we built a preliminary main-chain model. A subsequent search of structural databases, including AlphaFold predictions, allowed us to identify this density as the protein YbjP. It was only after this identification that we became aware of the related preprint by Horne et al. on BioRxvi (Posted March 19, 2025).

Therefore, our structural determination of YbjP was conducted entirely independently. We fully acknowledge and respect the work by Horne et al. and have already cited their reprint in our manuscript. While their detailed structural data, maps, and coordinates are not yet publicly available, we have described their findings appropriately. We agree that our manuscript can better reflect this context and will carefully check for any missing citations to ensure that their contribution is properly and clearly acknowledged.

We also believe that the two studies are mutually complementary and collectively reinforce the emerging understanding of YbjP.

Several results presented in the TolC-YbjP section do not represent new findings regarding TolC structure itself.

We agree that the TolC features we describe are consistent with previously reported structural characteristics. However, these observations could only be confirmed in the context of the newly determined TolC–YbjP subcomplex, which was not available prior to this study. We will clarify this point in the revision to avoid overstating novelty.

The structure and gating behaviour of TolC should be more thoroughly introduced in the Introduction, including prior work describing channel opening and conformational transitions.

We appreciate this suggestion and agree that a more comprehensive overview of TolC gating and conformational transitions will strengthen the Introduction. We will revise the text to incorporate relevant prior structural and functional studies.

The current manuscript does not discuss the mechanistic role of helices H3/H4 and H7/H8 in channel dilation, despite implying that YbjP binding may influence these features.

Thank you for this comment. The primary novel contributions of this manuscript are the identification of YbjP and the structural characterization of AcrB in three distinct states. The discussion of the dilation mechanism, while included because we observed the closed TolC-YbjP state, is a secondary point. In the revised manuscript, we will expand this discussion as suggested.

Only the original closed TolC structure is cited, and the manuscript does not address prior mutational studies involving the D396 region, though this residue is specifically highlighted in the presented structures.

We appreciate the reviewer drawing attention to this oversight. We will add citations to the relevant mutational and mechanistic studies, including those involving the D396 region, and more clearly discuss these findings in relation to our structural observations.

The manuscript provides only a general structural alignment between the closed TolC-YbjP subcomplex and the open TolC observed in the full pump assembly. However, multiple open, closed, and intermediate conformations of AcrAB-TolC have already been reported. Thus, YbjP alone cannot be assumed to account for TolC channel gating. A systematic comparison with existing structures is necessary to determine whether YbjP contributes any distinct allosteric modulation.

We agree with the reviewer’s assessment and appreciate the constructive suggestion. In our revised manuscript, we will expand the structural comparison to include previously reported open, closed, and intermediate AcrAB–TolC conformations. This expanded analysis will more clearly position our findings within the existing structural framework.

The analysis of AcrB peristaltic action is superficial, poorly substantiated and importantly, not novel. Several references to the ATP-synthase cycle have been provided, but this has been widely established already some 20 years ago - e.g. https://www.science.org/doi/10.1126/science.1131542.

We thank the reviewer for this comment. We fully acknowledge the foundational studies that established the AcrB functional cycle and its analogy to the ATP-synthase mechanism. While previous work indeed defined the LTO (Loose, Tight, Open) cycle of AcrB, those structures were obtained using AcrB in isolation. In contrast, our endogenous sample, which includes the native constraints of AcrA from above and the presence of AcrZ, reveals conformational changes in the transmembrane and porter domains that differ from those previously reported. We interpret these differences as reflecting a more physiologically relevant mechanism. In our revision, we will provide a detailed discussion to contextualize these distinctions within the existing literature.

The most significant limitation of the study is the absence of functional characterization of YbjP in vivo or in vitro. While the structural association between YbjP and TolC is interesting, the biological role of YbjP remains unclear.

We agree that the lack of functional characterization is a limitation of the present work. Our study focuses on structural elucidation and structural analysis. Although the recent preprint you mentioned suggests that YbjP deletion may not produce a strong phenotype, we are still interested in conducting additional experiments to explore its potential roles in future work. We will revise the text to clearly acknowledge this limitation.

Moreover, the manuscript does not examine structural differences between the presented complex and previously solved AcrAB-TolC or MexAB-OprM assemblies that might support a mechanistic model.

We thank the reviewer for this suggestion. We will incorporate a more detailed comparative analysis with existing AcrAB–TolC and MexAB–OprM structures and highlight similarities and differences that may inform mechanistic interpretation.