Edge-strand of BepA interacts with immature LptD on the β-barrel assembly machine to direct it to on- and off-pathways
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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, and 4, Figure 1-figure supplements 2, 3, and 4, Figure 2-figure supplements 1, and 2, Figure 3-figure supplements 1, and 2, and Figure 4-figure supplements 1, 2, and 3.
Article and author information
Author details
Funding
Japan Society for the Promotion of Science (18H06047)
- Ryoji Miyazaki
Japan Society for the Promotion of Science (19K21179)
- Ryoji Miyazaki
Japan Society for the Promotion of Science (20K15715)
- Ryoji Miyazaki
Japan Society for the Promotion of Science (15H01532)
- Yoshinori Akiyama
Japan Society for the Promotion of Science (18H023404)
- Yoshinori Akiyama
Institute for Fermentation, Osaka (Y-2020-02-027)
- Ryoji Miyazaki
Nagase Science Technology Foundation
- Yoshinori Akiyama
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Miyazaki et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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