Daimon, Y. | Search bioRxiv

bioRxiv2026

Structural and functional insights into multiple BAM-bound conformations of BepA enabling substrate triage at the outer membrane

The outer membrane (OM) of Gram-negative bacteria acts as a selective permeability barrier against toxic compounds, and for this function, requires proper assembly of outer membrane proteins (OMPs) by the {beta}-barrel assembly machinery (BAM) complex. A periplasmic metalloprotease BepA promotes maturation of LptD, an essential OMP, while degrades LptD intermediates aberrantly stalled at BAM. However, how BepA switches between these functions remains unclear. Here, we report cryo-EM structures of BAM- BepA complexes that capture multiple BepA conformations. In vivo crosslinking and cysteine-accessibility analyses show that possible regulatory elements, 6- and 9-loops that cover the proteolytic active site, can assume open conformations in living cells. Functional analyses reveal that 6-loop opening supports substrate interaction, degradation, and membrane association, whereas 9-loop opening is required for proteolytic activation and stabilizes BAM association. These findings provide insights into how distinct loop rearrangements regulate BepA-mediated substrate triage at BAM.

molecular biology↗

bioRxiv2020

Reversible auto-inhibitory regulation of Escherichia coli metallopeptidase BepA for selective β-barrel protein degradation

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD, but the underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features, in particular the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has a potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of the BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of the protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗