Edge-strand of BepA interacts with immature LptD on the β-barrel assembly machine to direct it to on- and off-pathways

  1. Ryoji Miyazaki
  2. Tetsuro Watanabe
  3. Kohei Yoshitani
  4. Yoshinori Akiyama

  • Curated by eLife

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    Evaluation Summary:

    This study reports a pivotal role of the conserved edge-strand of the periplasmic metalloprotease BepA in the folding and quality control of an outer membrane protein (OMP), the lipopolysaccharide transporter LptD, in gram-negative bacteria. The authors have discovered that, in addition to proteolytic activity, BepA has a chaperone-like activity and that BepA-mediated quality control of LptD and likely other OMPs occurs during their membrane insertion at the barrel assembly machinery. This result provides new insights into the biogenesis mechanisms of bacterial OMPs and the maintenance of OM integrity.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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Abstract

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of LptD, a β-barrel protein component of the lipopolysaccharide translocon. BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions probably on the β‐barrel assembly machinery (BAM) complex. However, how BepA recognizes and directs an immature LptD to different pathways remains unclear. Here, we explored the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge-strand located adjacent to the active site with LptD was crucial not only for proteolysis but also, unexpectedly, for assembly promotion of LptD. Site-directed crosslinking analyses indicated that the unstructured N-terminal half of the β-barrel-forming domain of an immature LptD contacts with the BepA edge-strand. Furthermore, the C-terminal region of the β-barrel-forming domain of the BepA-bound LptD intermediate interacted with a ‘seam’ strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the functional mechanism of BepA.

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  1. Version published to 10.7554/elife.70541 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    [...] Authors' rigorous experimental design (based on bacterial genetics and structural biology), solid biochemical assays (including photo-crosslinking, cysteine crosslinking, and Western blotting), and carefully drawn interpretation and conclusions are impressive. Finally, authors delineate the mechanisms of BepA activation and LptD biogenesis, which are supported by the current and previous studies by the authors and other research groups.

    Thank you for the nice summarization and the positive evaluation of our study.

    While this is overall a wonderful piece of work, this manuscript would be further improved by clarifying the following points:

    1. Authors examined how mutations (Pro and Cys scanning) on the edge-strand of BepA affected degradation and maturation of LptD.

    It was assumed that these mutations impact the structure of BepA only locally. However, a mutational effect can be propagated in an unexpected way affecting the structural integrity of other regions. Although authors tested that A106P retains proteolytic activity as shown by self-cleavage, a similar test (for example, in vitro experiments using a structureless substrate) may need to be extended to other mutations to support the conclusions.

    Thank you for the suggestion. Unfortunately, we have not succeeded in reproducibly detecting the proteolytic activity of BepA with purified BepA even when an unstructured substrate (-casein) is used and the assay was conducted at an elevated temperature, possibly because the protases activity of isolated BepA is tightly repressed by the mechanism that included His-246-mediated regulation as described in our paper (please see Introduction). Although BepA mutants with a mutation of His-246 or a deletion of H9 loop (these mutations release the His-246-mediated repression) significantly degrade -casein, a combination of these mutations with the edge-strand mutations should make the interpretation of the results complicated. We thus think that the suggested experiments cannot be conducted soon.

    Instead, we described the following points in the revised manuscript. Although we mentioned the self-cleavage activity of only the A106P mutant in the original manuscript, our results showed that the other edge-strand Pro mutants (other than F107P) exhibited significant self-cleavage activities as well (Figure 1-figure supplement 2B). In addition, the Pro mutants other than the A106P mutant degraded mis- or un-folded BamA at a detectable level (Figure 1-figure supplement 2A). Furthermore, all the Pro mutants accumulated at a level comparable to that of wild-type BepA. These observations together indicate that most of the Pro mutations specifically affected the edge-strand structure, but not drastically altered the active site or the protein's overall structures. We described the above points in the revised text (line 174 in p7 to line 181 in p8).

    1. In the result (Line 159), authors report chaperone-like activity of BepA. Here, the term "chaperone-like" is rather obscure regarding whether this activity facilitates LptD maturation without proteolysis (i.e., via holdase activity), or involves proteolysis as a part of quality control mechanisms. In another experiment, authors show that the chaperone-like activity may not necessarily involve proteolysis. It would be good to describe a possible molecular principle of how the edge-stand binding to the substrate can lead to chaperone activity.

    We suppose that the interaction of BepA (via the edge-strand) with an assembly intermediate of LptD on the BAM complex stabilizes the partially unfolded assembly intermediate of LptD on the BAM complex to help the association of LptE with LptD. This was explained in Discussion (lines 388–392 in p16) and the legend to Figure 5.

    Reviewer #2 (Public Review):

    The authors found that a conserved β-strand (edge-strand β2 of BepA) directly contacts with the N-terminal half of the β-barrel-forming domain of an immature LptD; the C-terminal region of the β-barrel-forming domain of the BepA-bound LptD intermediate interacts with a "seam" strand of BamA in the BAM complex. By combining crosslinking and mutational studies, they showed that the edge-strand of BepA may have both the proteolytic and the chaperone-like functions. Based on the authors' previous studies of BepA, they proposed a model that the edge-strand and His switch of BepA regulate BepA in LptD assembly and degradation.

    Thank you for the nice summarization of our study.

    Reviewer #3 (Public Review):

    [...] By performing an impressive systematic cross linking analysis, combined with previous known findings, the authors are able to dissect the general architecture of how BepA interacts with beta-barrel substrates as they are being assembled by the Bam complex. The experiments presented are nicely executed and the data are of high quality. I am convinced that the edge strand of BepA interacts with LptD, likely as it is assembling on the Bam complex. It is also clear that this interaction is functionally important because mutations in this region that disrupt the BepA-LptD interaction interfere with LptD maturation and degradation. This suggests that the substrate binding to the protease domain of BepA is important for both its chaperone and proteolytic activity. The work is well executed and will be of value to others interested in the regulation of membrane protein folding and, more broadly, in the biogenesis of the bacterial cell envelope.

    Thank you for the nice summarization and the positive evaluation of our study.

    While the authors conclusively establish a link between this region of BepA and its function, the data do not explain the underlying mechanism of how BepA discriminates between substrates targeted for integration into the membrane and those targeted for destruction. The model proposed at the end incorporates the presence of the edge strand of BepA, but its role in the process remains vague. As mentioned in the discussion, the mechanisms that control the switch from chaperone to protease function in BepA is likely governed by the loops that gate access to the catalytic residues proximal to the edge strand. It is possible that the edge strand may just be reporting on substrate binding to the protease domain active site. While this may be important for substrate recognition, it does not mean that the edge strand-substrate interaction plays a deterministic role in subsequent protein triage during LptD assembly.

    Our data demonstrated that the edge-strand of BepA directly binds a substrate. As pointed out by the reviewer, the involvement of the edge-strand in substrate binding has been known for other proteases. However, it was not known whether the substrate binding at the edge-strand contributes to the chaperone-like function; it was possible that the binding sites of a substrate on BepA during its proteolysis and its maturation are totally different as the chaperone-like activity of BepA is independent of its protease activity (it was conceivable, for example, that substrate binding during it maturation occurs on the surface of the C-terminal TPR domain that has been shown to interact with LptD）. Our results showed that the defective binding of a substrate (LptD) at the edge-strand impairs not only its proteolysis but also its normal maturation (assembly). Because the edge-strand-bound substrate would be directly presented to the proteolytic active site for its degradation, this binding step should be important for the determination of the fates of the bound substrate. Our results strongly suggest that the substrate binding by the edge-strand is a crucial common step required for the subsequent protein triage during the LptD assembly.

  3. eLife

    Evaluation Summary:

    This study reports a pivotal role of the conserved edge-strand of the periplasmic metalloprotease BepA in the folding and quality control of an outer membrane protein (OMP), the lipopolysaccharide transporter LptD, in gram-negative bacteria. The authors have discovered that, in addition to proteolytic activity, BepA has a chaperone-like activity and that BepA-mediated quality control of LptD and likely other OMPs occurs during their membrane insertion at the barrel assembly machinery. This result provides new insights into the biogenesis mechanisms of bacterial OMPs and the maintenance of OM integrity.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This work proposes a new mechanism for the biogenesis of the lipopolysaccharide transporter LptD in the bacterial outer membranes. Authors find that BepA, a periplasmic metalloprotease, plays a dual role, that is, facilitating maturation of the OMP as a chaperone and degrading its misassembled forms as a protease. In this mechanism, the edge strand of BepA mediates substrate binding and proteolytic activity. It is a novel finding that the OMP biogenesis can involve the formation of a ternary complex composed of the barrel assembly machinery (BAM), LptD and BepA, implying that BAM serves as a dynamic platform in which OMP substrates and quality control enzymes/chaperones are recruited.

    Authors' rigorous experimental design (based on bacterial genetics and structural biology), solid biochemical assays (including photo-crosslinking, cysteine crosslinking, and Western blotting), and carefully drawn interpretation and conclusions are impressive. Finally, authors delineate the mechanisms of BepA activation and LptD biogenesis, which are supported by the current and previous studies by the authors and other research groups.

    While this is overall a wonderful piece of work, this manuscript would be further improved by clarifying the following points:

    1. Authors examined how mutations (Pro and Cys scanning) on the edge-strand of BepA affected degradation and maturation of LptD.

    It was assumed that these mutations impact the structure of BepA only locally. However, a mutational effect can be propagated in an unexpected way affecting the structural integrity of other regions. Although authors tested that A106P retains proteolytic activity as shown by self-cleavage, a similar test (for example, in vitro experiments using a structureless substrate) may need to be extended to other mutations to support the conclusions.

    1. In the result (Line 159), authors report chaperone-like activity of BepA.

    Here, the term "chaperone-like" is rather obscure regarding whether this activity facilitates LptD maturation without proteolysis (i.e., via holdase activity), or involves proteolysis as a part of quality control mechanisms. In another experiment, authors show that the chaperone-like activity may not necessarily involve proteolysis. It would be good to describe a possible molecular principle of how the edge-stand binding to the substrate can lead to chaperone activity.

  5. eLife

    Reviewer #2 (Public Review):

    The authors found that a conserved β-strand (edge-strand β2 of BepA) directly contacts with the N-terminal half of the β-barrel-forming domain of an immature LptD; the C-terminal region of the β-barrel-forming domain of the BepA-bound LptD intermediate interacts with a "seam" strand of BamA in the BAM complex. By combining crosslinking and mutational studies, they showed that the edge-strand of BepA may have both the proteolytic and the chaperone-like functions. Based on the authors' previous studies of BepA, they proposed a model that the edge-strand and His switch of BepA regulate BepA in LptD assembly and degradation.

  6. eLife

    Reviewer #3 (Public Review):

    This study aims to understand how the regulatory mechanisms governing outer membrane protein biogenesis. Specifically, it focuses on the role of BepA during the biogenesis of the essential beta-barrel membrane protein, LptD.

    By performing an impressive systematic cross linking analysis, combined with previous known findings, the authors are able to dissect the general architecture of how BepA interacts with beta-barrel substrates as they are being assembled by the Bam complex. The experiments presented are nicely executed and the data are of high quality. I am convinced that the edge strand of BepA interacts with LptD, likely as it is assembling on the Bam complex. It is also clear that this interaction is functionally important because mutations in this region that disrupt the BepA-LptD interaction interfere with LptD maturation and degradation. This suggests that the substrate binding to the protease domain of BepA is important for both its chaperone and proteolytic activity. The work is well executed and will be of value to others interested in the regulation of membrane protein folding and, more broadly, in the biogenesis of the bacterial cell envelope.

    While the authors conclusively establish a link between this region of BepA and its function, the data do not explain the underlying mechanism of how BepA discriminates between substrates targeted for integration into the membrane and those targeted for destruction. The model proposed at the end incorporates the presence of the edge strand of BepA, but its role in the process remains vague. As mentioned in the discussion, the mechanisms that control the switch from chaperone to protease function in BepA is likely governed by the loops that gate access to the catalytic residues proximal to the edge strand. It is possible that the edge strand may just be reporting on substrate binding to the protease domain active site. While this may be important for substrate recognition, it does not mean that the edge strand-substrate interaction plays a deterministic role in subsequent protein triage during LptD assembly.

  7. Version published to 10.1101/2021.03.28.437416v2 on bioRxiv
  8. Version published to 10.1101/2021.03.28.437416v1 on bioRxiv