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

Bryant, Jack A.; Cadby, Ian T.; Chong, Zhi-Soon; Boelter, Gabriela; Sevastsyanovich, Yanina R.; Morris, Faye C.; Cunningham, Adam F.; Kritikos, George; Meek, Richard W.; Banzhaf, Manuel; Chng, Shu‐Sin; Lovering, A.L.; Henderson, Ian R.

doi:10.1128/jb.00434-20

Cited by 9 publications

(17 citation statements)

References 55 publications

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“…We note that during the preparation of this manuscript, an archived preprint (35) reported determination of the E. coli BepA structure and analysis of His-246. The structure solved in that study was very similar to the structure we have reported previously and, consistent with our results, their mutational analysis of His-246 and the α9/H246 loop in that study reportedly supported the importance of these elements in the overall BepA function, although the protease activity of the His-246 mutants of BepA was not directly examined in that report.…”

Section: Discussionmentioning

confidence: 99%

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

Daimon

Narita

Miyazaki

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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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. 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 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 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 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.

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Section: Discussionmentioning

confidence: 99%

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

Daimon

Narita

Miyazaki

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…A Dali search of the PDB with the structure of NleC (PDB id 4Q3J) revealed the E. coli BepA metalloprotease (PDB id 6SAR, [ 97 ]) as its closest, no-T3SE relative (Z-score = 5.3 and rmsd = 3.5 Å). A noticeable difference between the proteins is the distortion of a Ψ-loop β-sheet motif in the T3SE [ 98 ].…”

Section: Marginal Resemblance Of T3se Folds To Functionally Related Proteinsmentioning

confidence: 99%

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles

Gazi

Kokkinidis

Fadouloglou

2021

IJMS

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Type III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell’s metabolism for the benefit of the bacterium to bypassing the host’s defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains.

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“…The edge strand directly interacts with a substrate polypeptide and converts it into an extended conformation for its presentation to the active site and proteolysis. The solved structures of Escherichia coli BepA (24, 25) show that it has a β-strand (β2) that is conserved among the M48-peptidases and is located adjacent to the active site (Fig.1 A and Fig. S1), suggesting that this strand presumably acts as an edge strand.…”

Section: Resultsmentioning

confidence: 99%

“…This finding was unexpected as it raised a question of how degradation of the normally assembling LptD intermediate can be avoided despite its interaction with the edge strand near the protease active site. In the BepA structures, the conserved His-246 residue is coordinated to a zinc ion in the active site to block the activation of a water molecule necessary for the catalysis of the proteolytic reaction (24, 25). We have recently reported that His-246 acts as a switch to regulate the proteolytic activity of BepA (27).…”

Section: Discussionmentioning

confidence: 99%

“…The BepA protein consists of an N-terminal M48 metallopeptidase domain and a C-terminal tetratricopeptide repeat (TPR) domain that are associated closely to form a compact structure (Fig. 1 A ) (24, 25). Our previous study suggested that the TPR domain of BepA directly interacts with LptD and with the BAM complex with its TPR domain inserted into the interior of the periplasmic part (brim) of the BAM complex (20, 26).…”

Section: Introductionmentioning

confidence: 99%

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

Miyazaki

Watanabe

Yoshitani

et al. 2021

Preprint

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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.

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

Cited by 9 publications

References 55 publications

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

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

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles

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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