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.

The crystal structure of CbpD clarifies substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases

“…The -strand interaction observed between the GbpA2 domains of crystallographic symmetry mates raises another question about the possible biological function of structural features of CbpD. Edge -strands are well known to mediate protein-protein interactions, which in some cases are dynamic and interchangeable (Richardson & Richardson, 2002;Monteiro et al, 2013;Yu et al, 2014;Miyazaki et al, 2021). Given the broad substrate repertoire of the P. aeruginosa T2SS (Cianciotto & White, 2017) and the expectation that the recognition of secreted proteins by multiple proteins along the secretion-machinery pathway relies on dynamic, short-lived interactions (Douzi et al, 2011;Michel-Souzy et al, 2018) as well as inherent disorder (Gu et al, 2017;Pineau et al, 2021), it is interesting to imagine that the 15 edge strand of CbpD may serve as a handle for interaction with secretion-machinery proteins along the pathway through the cell envelope.…”

CbpD crystal structure adds intrigue to substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases

“…The β-strand interaction observed between GbpA2 domains of crystallographic symmetry mates raises another question about the possible biological function of structural features of CbpD. Edge β-strands are well-known to mediate protein:protein interactions, which in some cases are dynamic and interchangeable (Miyazaki et al ., 2021; Richardson & Richardson, 2002; Monteiro et al ., 2013; Yu et al ., 2014). Given the broad substrate repertoire of the P. aeruginosa T2SS (Cianciotto & White, 2017), and the expectation that recognition of secreted proteins by the multiple proteins along the secretion machinery pathway relies on dynamic, short-lived interactions (Douzi et al ., 2011; Michel-Souzy et al ., 2018) as well as inherent disorder (Gu et al ., 2017; Pineau et al ., 2021), it is interesting to imagine that the β15 edge strand of CbpD may serve as a handle for interaction with secretion machinery proteins along the pathway through the cell envelope.…”

Analyzing protein intermediate interactions in living E. coli cells using site-specific photo-crosslinking combined with chemical crosslinking

“… 9 pEVOL-pBpF is available from Addgene. pNB91 (pMW118- bepA(E137Q) ), a plasmid encoding BepA carrying the proteolytic active site substitution, E137Q) 10 ; the substitution would stabilize the interaction between BepA and LptD. 11 pRM829 (pRM294- lptD(E733C)-his 10 ), 10 a plasmid encoding the E733C mutant form of LptD-His 10 (LptD with a C-terminal decahisitidine tag) for BMB-crosslinking with the chromosomally encoded BamA protein bearing an S439C substitution.…”

“… pNB91 (pMW118- bepA(E137Q) ), a plasmid encoding BepA carrying the proteolytic active site substitution, E137Q) 10 ; the substitution would stabilize the interaction between BepA and LptD. 11 pRM829 (pRM294- lptD(E733C)-his 10 ), 10 a plasmid encoding the E733C mutant form of LptD-His 10 (LptD with a C-terminal decahisitidine tag) for BMB-crosslinking with the chromosomally encoded BamA protein bearing an S439C substitution. CRITICAL: The recovery of the crosslinked products was much lower with shorter His-tags such as the widely-used hexa-His tag because the binding affinity of the decahistidine tag to the Ni-NTA agarose is higher than that of the hexahistidine tag.…”

“… CRITICAL: The recovery of the crosslinked products was much lower with shorter His-tags such as the widely-used hexa-His tag because the binding affinity of the decahistidine tag to the Ni-NTA agarose is higher than that of the hexahistidine tag. pRM831(pRM294- lptD(Y331amb/E733C)-his 10 ), 10 a plasmid encoding LptD-His 10 carrying both the E733C substitution for the BMB-crosslinking with chromosomally encoded BamA(S439C) and the Y331 amb substitution for photo-crosslinking with BepA. RM3655 (Δ pro-lac thi /F′ lacI q Z Δ M15 Y + pro + Δ bepA bamA(S439C) zae-502 ::Tn 10 ), 10 an E. coli strain carrying a chromosomal bamA(S439C) mutant gene for BMB-crosslinking with LptD.…”