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.

No abstract

The outer membrane (OM) of gram-negative bacteria is crucial for maintenance of cell viability as it 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 an essential OM protein, LptD, a β-barrel component of the lipopolysaccharide translocon. We have previously shown that 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 possibly on the β‐barrel assembly machinery (BAM) complex. However, the mechanistic details of how BepA recognizes and directs the LptD assembly intermediates to different pathways remains unclear. Here, we performed site-directed mutagenesis and crosslinking experiments to explore 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 for assembly promotion of LptD. Site-directed crosslinking analysis indicated that the unstructured N-terminal half of the β-barrel-forming domain of an LptD assembly intermediate directly 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 involvement of BepA in the maintenance of OM structure and function, which can be helpful in developing OM-targeted novel drugs.