Reversible auto-inhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation

Abstract

<jats:title>Abstract</jats:title><jats:p><jats:italic>Escherichia coli</jats:italic> 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 <jats:italic>in vivo</jats:italic> and <jats:italic>in vitro</jats:italic>. 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.</jats:p>

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

“…This species probably represents a “normal” assembly intermediate as it is associated with the BAM complex (See below) and can be converted to the mature form (LptD NC ) when LptE is co-expressed (31). As reported previously (27, 32), overproduced LptD was degraded by co-expressed wild-type BepA to generate discrete degradation products (Fig. 1 B ).…”

“…We then investigated the effects of β2 mutations on the proteolytic activity of BepA against overproduced LptD. When LptD is overproduced from a multi-copy plasmid, it mainly accumulates in the form of LptD C possibly due to the limited availability of its partner protein, LptE (26, 27). This species probably represents a “normal” assembly intermediate as it is associated with the BAM complex (See below) and can be converted to the mature form (LptD NC ) when LptE is co-expressed (31).…”

“…A mutational study has suggested that these interactions are important for BepA functions (24). Recent study have also shown that the H246 residue of BepA that coordinates the zinc ion at the proteolytic active site acts as an ON/OFF switch (His switch) for the proteolytic activity of BepA (25, 27). The dual functions of BepA should be appropriately regulated because the unregulated expression of the proteolytic activity of BepA caused by an H246 mutation leads to the degradation of the normally assembling LptD intermediate (27).…”

“…Recent study have also shown that the H246 residue of BepA that coordinates the zinc ion at the proteolytic active site acts as an ON/OFF switch (His switch) for the proteolytic activity of BepA (25, 27). The dual functions of BepA should be appropriately regulated because the unregulated expression of the proteolytic activity of BepA caused by an H246 mutation leads to the degradation of the normally assembling LptD intermediate (27). However, information on the molecular mechanism of this regulation and the modes of the BepA-LptD interaction in each BepA function remains elusive.…”

Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA

“…Through the use of disulfide bond tethering, we also demonstrate that the plug must be mobile for proteolytic activity of BepA on the in vivo substrate BamA. We note that while this study was under review, similar work was presented in a preprint report revealing that residue H246 is important for BepA function ( 44 ) and that the active-site plug can be tethered using the identical residues reported here, leading to switchable proteolytic activity on the LptD substrate and the in vitro substrate α-casein. This confirms our results and suggests that the plug may act in an autoregulatory fashion and must relocate to facilitate access for the substrate and subsequent proteolytic activity.…”