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

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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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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.

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Ryoji Miyazaki

Kyoto University, Kyoto, Japan

Competing interests
The authors declare that no competing interests exist.
Tetsuro Watanabe

Kyoto University, Kyoto, Japan

Competing interests
The authors declare that no competing interests exist.
Kohei Yoshitani

Kyoto University, Kyoto, Japan

Competing interests
The authors declare that no competing interests exist.
Yoshinori Akiyama

Kyoto University, Kyoto, Japan

For correspondence
yakiyama@infront.kyoto-u.ac.jp

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-4483-5408

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.

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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.