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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

  1. Ryoji Miyazaki
  2. Tetsuro Watanabe
  3. Kohei Yoshitani
  4. Yoshinori Akiyama  Is a corresponding author
  1. Institute for Frontier Life and Medical Sciences, Kyoto University, Japan
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Cite this article as: eLife 2021;10:e70541 doi: 10.7554/eLife.70541

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.

Introduction

The cell envelope of diderm bacteria is composed of two membranes, namely the inner (cytoplasmic) membrane (IM) and the outer membrane (OM). The intermembrane space, known as periplasmic space, contains a peptidoglycan layer. The OM is the outermost layer of a cell directly facing the external milieu and acts as a selective permeability barrier that prevents the penetration of toxic compounds including antibiotics (Nikaido, 2003). The cell surface localization as well as the functional importance of the OM make its components suitable drug targets.

Outer membrane proteins (OMPs), generally exhibiting a β-barrel fold formed by more than eight β-strands, play important roles in maintaining the structural and functional integrity of the OM (Konovalova et al., 2017). Therefore, irregularities in OMP biogenesis result in elevated cellular sensitivity to toxic compounds (Hart et al., 2019; Oh et al., 2011). After synthesis in the cytoplasm and following translocation across the IM to the periplasm through the SecYEG translocon, OMPs are delivered to the OM by periplasmic chaperones such as DegP, Skp, and SurA, and are finally integrated into the OM (Konovalova et al., 2017; Plummer and Fleming, 2016; Ricci and Silhavy, 2019). The OM assembly of OMPs is mediated by the β-barrel assembly machinery (BAM) complex consisting of a β-barrel OMP, BamA, and four lipoproteins, namely BamB, BamC, BamD, and BamE (Plummer and Fleming, 2016; Ricci and Silhavy, 2019; Tomasek and Kahne, 2021). Among the BAM complex subunits, BamA and BamD are the only essential components, although recent studies have shown that certain BamA mutations render all other BAM subunits dispensable (Hart et al., 2020). The BAM complex has a silk-hat-like structure (Bakelar et al., 2016; Gu et al., 2016; Han et al., 2016; Iadanza et al., 2016; Tomasek et al., 2020); the OM-embedded C-terminal β-barrel domain of BamA forms the ‘crown’ and the N-terminal periplasmic polypeptide transport-associated (POTRA) domains of BamA form the ‘brim’ together with the BamB/C/D/E lipoproteins.

Lipopolysaccharide (LPS), another major OM constituent localized in the outer leaflet of the OM, is also important for the maintenance of the structure and function of the OM (Sperandeo et al., 2017). LPS is synthesized on the cytoplasmic side of the IM and flipped across the IM to the periplasm. After maturation, it is transported to the OM by the LPS transport (Lpt) proteins (Sperandeo et al., 2017). A heterodimer of LptD, a β-barrel OMP, and LptE, a lipoprotein, plays roles in the final step to insert LPS into the OM (Dong et al., 2014; Qiao et al., 2014; Wu et al., 2006). The OM assembly process of LptD is unique in that it is accompanied by the rearrangement of intramolecular disulfide bonds. The mature form of LptD possesses two disulfide bonds formed by non-consecutive pairs of Cys residues (C31–C724 and C173–C725) (Ruiz et al., 2010). It has been shown, however, that an assembly intermediate of LptD having disulfide bonds formed by consecutive pairs of the Cys residues (C31–C173 and C724–C725) (LptDC; LptD with Consecutive disulfide bods) is first generated and isomerized to LptDNC (LptD with Non-Consecutive disulfide bonds) during its assembly/maturation, which is triggered by the association of LptD with LptE (Chng et al., 2012; Narita et al., 2013). The LptDC to LptDNC conversion should occur at a later step in the OM assembly because LptD presumably associates with LptE on the BAM complex (Chimalakonda et al., 2011; Chng et al., 2012; Lee et al., 2016; Narita et al., 2013).

BepA (formally called YfgC), a bi-functional periplasmic protein that plays an important role in maintaining OM integrity (Narita et al., 2013), belongs to the M48 family zinc-metallopeptidases that include prokaryotic and eukaryotic proteases (such as Ste24, Oma1, and HptX) involved in membrane quality control (Rawlings et al., 2018). We have previously shown that BepA is involved in the biogenesis and quality control of LptD. While BepA promotes the LptDC to LptDNC conversion (chaperone-like function) (Narita et al., 2013), it also degrades the stalled or misassembled LptDC molecules that are generated due to an lptD mutation (lptD4213) or decreased availability of or weakened interaction with LptE (protease function) (Narita et al., 2013; Soltes et al., 2017). BepA also degrades BamA whose assembly/folding has been impaired in the absence of a periplasmic chaperone, SurA (Daimon et al., 2017), suggesting that BepA can also act in quality control of some other OM proteins. 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 (Figure 1A; Bryant et al., 2020; Shahrizal et al., 2019). 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 (Daimon et al., 2017; Narita et al., 2013). A mutational study has suggested that these interactions are important for BepA functions (Daimon et al., 2017). Recent studies have also shown that the His-246 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 (Bryant et al., 2020; Daimon et al., 2020). 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 (Daimon et al., 2020). However, information on the molecular mechanism of this regulation and the modes of the BepA–LptD interaction in each BepA function remains elusive.

Figure 1 with 4 supplements see all
The edge-strand of BepA is crucial for functional interaction with LptD.

(A) Crystal structure of BepA (PDB code: 6AIT). The peptidase and the TPR domains of BepA are shown in gray and orange, respectively. The edge-strand, the proteolytic active site (the HExxH motif and the third zinc ligand, Glu-201), and the regulatory His-246 residue (His switch) in the peptidase domain are shown in red, blue, and green, respectively, and the coordinated zinc atom is shown in yellow. An enlarged view of the active site region is shown in right. (B) Protease activities of the BepA edge-strand mutants. Cells of SN56 (ΔbepA) carrying pTWV228-lptD-his10 and either pSTD689 or pSTD689‐bepA plasmids were grown at 30°C in L-medium until early log phase and induced with 1 mM IPTG for 1 hr. Total cellular proteins were acid-precipitated and analyzed by 7.5 or 10% Laemmli SDS‐PAGE and immunoblotting with the indicated antibodies. (C) In vivo photo-crosslinking analysis of the BepA edge-strand. Cells of SN56 carrying pEVOL-pBpF and pUC18‐bepA(E137Q, amb)-his10 plasmids were grown at 30°C in L-medium containing 0.02% arabinose and 0.5 mM pBPA until early log phase, and induced with 1 mM IPTG for 1 hr to express the indicated BepA(pBPA) variants. The cultures were divided into two portions, each of which was treated with or without UV-irradiation for 10 min at 4°C. Proteins of the total membrane fractions were subjected to pull-down with Ni-NTA agarose. Purified proteins were analyzed by 7.5% Laemmli SDS‐PAGE and immunoblotting with the indicated antibodies. Open triangles indicate unknown crosslinked products. (D) Chaperone-like activities of the BepA edge-strand mutants. Cells of SN56 carrying pSTD689 or a pSTD689‐bepA plasmid were grown at 30°C in M9-based medium until early log phase, induced with 1 mM IPTG for 15 min, pulse-labeled with 35S-Met for 1 min and chased for the indicated periods. At each time point, total cellular proteins were acid-precipitated, subjected to IP with an anti-LptD antibody, and analyzed by 7.5% Laemmli SDS‐PAGE followed by phosphorimaging. The ratio of the band intensities of LptDNC at each time point to that of total LptD (LptDC+LptDNC) at 5 min was quantitated and the mean values were plotted with S.D. (n=2). The result shown is a representative of two independent experiments that were conducted using the same transformants (i.e., two technical replicates). See Figure 1—source data 1 for gel images and quantitated band intensities data for (D).

Figure 1—source data 1

A Zip file containing gel images.

(B–D) For the immunoblotting experiments using the anti-BepA and anti-LptD antibodies and quantified band intensity data for the pulse-chase experiments using the anti-LptD antibody.

https://cdn.elifesciences.org/articles/70541/elife-70541-fig1-data1-v2.zip

Here, we investigated the mechanism by which BepA established interaction with LptD in the promotion of its assembly and degradation. Our results showed that a conserved β-strand (edge-strand) located adjacent to the BepA active site directly contacts with LptD and plays important roles in substrate proteolysis, like many other proteases. In addition, we unexpectedly found that the edge-strand-mediated interaction with a substrate is also required for the chaperone-like function of BepA, which should be enabled by the His switch-mediated repression of the proteolytic activity. Crosslinking experiments demonstrated that BepA could interact with an LptD molecule assembling on the BAM complex. Based on these observations, we propose a model explaining the edge-strand and His switch-mediated functional regulation of BepA in LptD assembly/degradation.

Results

Interaction of the BepA edge-strand with LptD is crucial not only for proteolysis but also for assembly promotion of LptD by BepA

Zinc-metallopeptidases usually possess a β-strand, called edge-strand, located close to their proteolytic active sites (Akiyama et al., 2015; López-Pelegrín et al., 2013; Stöcker and Bode, 1995). While the edge-strand is known to play a critical role in substrate proteolysis by directly interacting with a substrate polypeptide by the strand addition mechanism and converts it into an extended conformation for its presentation to the active site and proteolysis, it has not been well characterized in M48 proteases. The solved structures of Escherichia coli BepA (Bryant et al., 2020; Shahrizal et al., 2019) show that it has a β-strand (β2) that is conserved among the M48-peptidases and is located adjacent to the active site (Figure 1A and Figure 1—figure supplement 1), suggesting that this strand presumably acts as an edge-strand. To examine the role of the β2-strand in BepA functions, we constructed BepA mutants by introducing Pro at each position in β2 (from Asn-105 to Phe-110; Figure 1A). 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 LptDC possibly due to the limited availability of its partner protein, LptE (Daimon et al., 2020; Daimon et al., 2017). 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 (LptDNC) when LptE is co-expressed (Miyazaki et al., 2018). As reported previously (Daimon et al., 2020; Daimon et al., 2017), overproduced LptD was degraded by co-expressed wild-type BepA to generate discrete degradation products (Figure 1B). To examine the possible roles of the β2-strand in the function of BepA, we introduced a Pro substitution into the β2-strand, as a Pro residue would affect the secondary structure, and thus the function, of this strand. We found that the expression of a few BepA mutants (N105P, A106P, F107P, and A108P) led to a significantly decreased generation of the LptD degradation products (Figure 1B). Furthermore, some of these mutations compromised the degradation of BamA in a ΔsurA strain (Daimon et al., 2017) and the self-cleavage of BepA-His10 (BepA possessing a C-terminal His10-tag) within the His10-tag (Narita et al., 2013; Figure 1—figure supplement 2A and B). These results strongly suggest that the β2-strand is important for the proteolytic activity of BepA.

We next analyzed whether the β2-strand directly interacts with a substrate by using a site-directed in vivo photo-crosslinking approach (Chin and Schultz, 2002; Miyazaki et al., 2020a). We expressed derivatives of BepA-His10 harboring a photoreactive amino acid analog, p-benzoyl-L-phenylalanine (pBPA), at each position in the β2-strand in a ΔbepA strain and examined their complementation activity regarding erythromycin (EM) sensitivity of the cell and self-cleavage of BepA-His10. The results showed that certain mutants (F107pBPA, F109pBPA, and F110pBPA) were as functional as the wild-type, but others exhibited neither significant complementation activity nor self-cleavage (Figure 1—figure supplement 3A and B). For the photo-crosslinking experiments conducted in this study, we used BepA variants harboring the E137Q mutation in the H136ExxH motif for two reasons. First, this mutation would repress the possible degradation of LptD by the BepA derivatives. Second, our previous study has shown that this mutation stabilizes the interaction of BepA with LptD (Daimon et al., 2017). Following UV-irradiation of cells expressing each of the BepA mutants, BepA-His10 and its crosslinked products were purified from the membrane fractions by affinity isolation using Ni-NTA agarose and subjected to immunoblotting analysis (Figure 1C). The N105pBPA, A106pBPA, F107pBPA, and A108pBPA derivatives of BepA generated, in a UV-dependent manner, evident crosslinked products that reacted with both anti-BepA and anti-LptD antibodies. Taken together, these results imply that the β2-strand of BepA directly interacts with LptD and plays an important role in their degradation. The structure, intramolecular disposition, as well as involvement in substrate interaction and proteolysis of the β2-strand strongly supports the hypothesis that it indeed acts as the edge-strand to recognize and present a substrate to the active site. We have thus referred to the β2-strand as the ‘edge-strand’ hereafter. BepA N105pBPA and F107pBPA derivatives also generated the unidentified crosslinked products (Figure 1C, open arrowheads), suggesting that the edge-strand would interact with some other substrates. The differential effects of the mutations on the degradation of LptD and BamA and the self-cleavage of the C-terminal tag (Figure 1B and Figure 1—figure supplement 2A and B) might reflect their different interaction properties and/or affinities for the BepA edge-strand. Our previous study also suggested substrate-specific interaction of an edge-strand for another metallopeptidase, RseP (Akiyama et al., 2015).

BepA not only degrades the stalled or misassembled LptD (when LptD is on the off-pathway) but also promotes the maturation of LptD through facilitating its assembly with the partner protein LptE (chaperone-like activity) (on the normal (on-) pathway) (Narita et al., 2013). Therefore, we examined the effects of the edge-strand Pro mutations on the chaperone-like activity of BepA. LptD possesses two intramolecular disulfide bonds. While the mature (fully assembled) LptD has non-consecutive disulfide bonds, an assembly intermediate form of LptD (LptDC) that accumulates in the absence of functional BepA possesses consecutive disulfide bonds. Consistent with our previous results (Narita et al., 2013), the expression of the wild-type BepA in a ΔbepA strain markedly decreased the accumulation of LptDC (Figure 1—figure supplement 4A). On the contrary, only a partial decrease in accumulation was observed with the expression of the E137Q mutant. We found that the A106P mutant was defective in reducing LptDC accumulation, suggesting that a mutation in the edge-strand could affect the chaperone-like function (Figure 1—figure supplement 4A). Subsequently, we examined the effects of two edge-strand mutations—A106P and F107P—that markedly compromised LptD degradation on the chaperone-like function of BepA using pulse-chase experiments. Pulse-chase analysis showed that LptD was largely stable, but received a slight degradation during the chase period under the condition used (Figure 1D, left panel, +ME). It is likely that some of LptDC was degraded by BepA during the chase period, because LptDNC is largely BepA-resistant (Daimon et al., 2020). Thus, the ratio of LptDNC to the total LptD (LptDC+LptDNC) at each chase point does not exactly reflect the efficiency of the LptDC-to-LptDNC conversion of pulse-labeled LptD. To examine only the chaperone-like activities (promotion of the LptDNC generation) without the possible effect of the LptD degradation by the residual proteolytic activities of the A106P and the F107P, the relative amounts of LptDNC at each chase point to the total LptD (LptDC+LptDNC) at an early chase point (5 min) were calculated and plotted as quantified data (Figure 1D, right panel). The expression of the wild-type BepA in ΔbepA cells significantly accelerated the conversion of LptDC to LptDNC, whereas the expression of the protease-dead E137Q mutant demonstrated weakened conversion (Figure 1D). The acceleration of the LptDC to LptDNC conversion by the F107P and the A106P mutants was much weaker than that by the wild-type BepA (almost the same as compared with the E137Q mutant) (Figure 1D). These results unexpectedly suggested that the edge-strand was also important for the chaperone-like activity of BepA.

We constructed BepA derivatives with a Cys substitution (note that BepA intrinsically possesses no Cys residue) at the position of Asn-105, Ala-106, or Phe-107 in the edge-strand to examine whether a specific residue in this strand is required for the BepA's functions. We chose a Cys substitution, because (i) the Cys mutants can be used in the disulfide crosslinking experiments described below, and (ii) a previous study strongly suggested that a Cys mutation does not affect the secondary structure of an edge-strand in another protease RseP (Akiyama et al., 2015). These BepA Cys constructs exhibited almost normal chaperone-like and proteolytic functions (Figure 1—figure supplement 4B and C). This supports the idea that the secondary structure of the edge-strand is more important than the individual amino acid residues for its function, although some contribution of the side-chains of the amino acids to the functions of the edge-strand cannot be excluded. Note that, although it is possible that a Pro mutation also affects the structures of the overall BepA protein and/or the active site around the edge-strand, the edge-strand Pro mutants (other than F107P) still exhibited significant self-cleavage of the probably unstructured-terminal tag (Figure 1—figure supplement 2B). In addition, the Pro mutants (other than A106P) degraded mis- or un-folded BamA at a detectable level (Figure 1—figure supplement 2B). Together with the result that these mutants accumulated at a level comparable to that of wild-type BepA, the above observations suggest that most of the Pro mutations specifically affected the edge-strand structure, but not drastically altered the active site or the protein's overall structures.

BepA interacts with the N-terminal half of the β-barrel-forming domain of the LptD assembly intermediate

While BepA interacts with LptD to promote either its proper OM assembly or proteolytic elimination depending on the situation (Narita et al., 2013), the details of the BepA–LptD interaction, including the region(s) in LptD to which BepA binds, remain largely unknown. Thus, we performed a systematic photo-crosslinking analysis to identify the BepA-contact region in the LptD assembly intermediate LptDC. We performed photo-crosslinking experiments in cells ectopically co-expressing an LptD derivative containing a photo-reactive amino acid analog pBPA [LptD(pBPA)] and a protease-dead variant of BepA, BepA(E137Q). We first introduced pBPA at each of the 50 positions (approximately every 15 residues) in the mature part of LptD and performed photo-crosslinking analysis. Cells expressing LptD(pBPA)-His10 and BepA(E137Q) were grown and UV-irradiated, and the total cellular proteins were analyzed by immunoblotting with anti-BepA and anti-His antibodies. Under this condition, expressed LptD-His10 was considerably accumulated as LptDC (Figure 2—figure supplement 1) irrespective of co-expression of BepA(E137Q). We detected clear crosslinking with BepA mainly in the N-terminal half of the LptD β-barrel-forming domain (Figure 2A). We then performed a detailed photo-crosslinking analysis for the 20 additional sites in the N-terminal half of the LptD β-barrel-forming domain (Figure 2B) and found that BepA was crosslinked at several of these sites. The residues at the BepA-cross-linkable sites were oriented both inward and outward in the mature LptD β-barrel (Figure 2C and D). Moreover, the residue Gln-393 at which the strongest crosslinking was observed was oriented inward. These results suggest that LptD while interacting with BepA would not assume a higher-order structure like closed β-barrel (see Discussion). We selected a few LptD(pBPA) derivatives that had been crosslinked with BepA as representatives and examined their functionality. They supported the growth of LptD-depleted cells when expressed from a plasmid, indicating that they were functional (Figure 2—figure supplement 2). The above-mentioned crosslinking results thus likely reflect a functional interaction of LptD with BepA in the normal assembly pathway.

Figure 2 with 2 supplements see all
Photo-crosslinking of the β-barrel forming domain of LptD with BepA.

(A, B) In vivo photo-crosslinking between LptD and BepA. Cells of RM2243 (bepA(E137Q)) carrying pEVOL-pBpF, pMW118‐bepA(E137Q), and pRM294-lptD(amb)-his10 plasmids were grown at 30°C in L-medium containing 0.02% and 0.5 mM pBPA until early log phase and induced with 1 mM IPTG for 3 hr to express the indicated LptD(pBPA) variants. The cultures were then divided into two portions, each of which was UV-irradiated for 10 min at 4°C. Total cellular proteins were acid-precipitated and analyzed by 7.5% Laemmli SDS‐PAGE and immunoblotting with the indicated antibodies. Most of the LptD mutants were accumulated in comparable amounts. LptD-His10xBepA crosslinked products were not detectable with an anti-His antibody due to its low reactivity to LptD-His10 in this and the following experiments. Amino acid residues shown in red and blue indicate the ones whose side chain is pointing inward and outward, respectively. Amino acid residues shown in green indicate the ones located in the loop regions. The result shown is a representative of two technical replicates. (C) Summary of the BepA crosslinked positions in LptD. Positions where the crosslinking with BepA was clearly and reproducibly detected are indicated by colored dots. (D) Mapping of the BepA crosslinked positions on the barrel domain of LptD in the Escherichia coli LptD–LptE structure (PDB code: 4RHB). LptD and LptE are shown in gray and light green, respectively. The N-terminal strand and the β-signal (C-terminal region) in the LptD β-barrel domain are shown in magenta and light blue, respectively. The top view of the LptD/E structure from extracellular space (upper) and the side view of the N-terminal region of LptD β-domain (lower) are shown. The positions where the crosslinking with BepA was observed were indicated by spheres colored as above. See Figure 2—source data 1 for gel images for (A, B).

Figure 2—source data 1

A Zip file containing gel images (A, B) for the immunoblotting experiments using the anti-BepA and anti-His tag antibodies.

https://cdn.elifesciences.org/articles/70541/elife-70541-fig2-data1-v2.zip

BepA edge-strand directly interacts with the Tyr-331 residue in the β7 strand of the LptD β-barrel domain

We investigated further to identify the region of LptD that interacts with the BepA edge-strand. First, we examined the effects of the BepA edge-strand Pro mutations (F107P and A106P) on the LptD(pBPA)–BepA crosslinking. The F107P mutation significantly decreased the efficiency of the crosslinking of BepA(N105pBPA) and BepA(A106pBPA) with LptD (Figure 3—figure supplement 1). Additionally, the A106P mutation exhibited a similar effect on the crosslinking of BepA(N105pBPA) with LptD. Based on these effects on crosslinking, we inferred that these mutations affected the interaction of the edge-strand with LptD. Subsequently, we selected several LptD(pBPA) derivatives that showed relatively strong crosslinking with BepA and examined the effect of F107P and A106P mutations in the BepA edge-strand on the crosslinking of LptD(pBPA) with BepA. We found that these mutations altered LptD–BepA crosslinking in a site-specific manner. Further, the amount of crosslinked products markedly decreased for LptD(Y331pBPA), but not for other mutants (Figure 3A). These results strongly suggest that the region around Tyr-331 in the β7 strand of the LptD β-barrel domain (Figure 2D) is crosslinked with the edge-strand of BepA. Note that, while we detected LptD-BepA crosslinked products ranging from 150 to 250 kDa with BepA derivatives having pBPA in the edge-strand (Figure 1C), we did not observe similar multiple crosslinked products with LptD(Y331pBPA) (Figure 2). The exact reason for this is unknown, but it might be ascribed to the crosslinking of pBPA in the BepA edge-strand to different LptD positions that are close spatially but distant in the primary sequence, which could generate crosslinked products with different mobility (i.e., different apparent sizes).

Figure 3 with 2 supplements see all
BepA edge-strand directly contacts with the Tyr-331 residue in the N-terminal half region of the LptD β-barrel forming domain.

(A) Effect of the BepA edge-strand mutations on the crosslinking between BepA and the LptD derivatives having pBPA in the N-terminal half region of the LptD β-barrel-forming domain. Cells of SN56 (ΔbepA) carrying pEVOL-pBpF, pMW118‐bepA(E137Q, mut), and pRM294-lptD(amb)-his10 were grown, induced to express a BepA and a LptDpBPA derivative, and subjected to photo-crosslinking analysis as described in Figure 2. (B) Disulfide crosslinking between the Cys residues in the edge-strand of BepA and the N-terminal half region of the LptD β-barrel-forming domain. Cells of SN56 (ΔbepA) carrying a combination of plasmids encoding WT or a Cys-introduced mutant of BepA and LptD-His10 as indicated were grown in L-medium and induced with 1 mM IPTG for 3 hr to express BepA(Cys) and LptD(Cys)-His10. Total cellular proteins were acid-precipitated, solubilized with SDS buffer containing NEM (for blocking free thiol groups), and subjected to pull-down with Ni-NTA agarose. The purified proteins were treated with or without 2-mercaptoethanol (ME) and analyzed by 7.5% Laemmli SDS-PAGE and immunoblotting with the indicated antibodies. The result shown is a representative of two technical replicates. See Figure 3—source data 1 for gel images for (A, B).

Figure 3—source data 1

A Zip file containing gel images (A, B) for the immunoblotting experiments using the anti-BepA and anti-His tag antibodies.

https://cdn.elifesciences.org/articles/70541/elife-70541-fig3-data1-v2.zip

To further confirm the direct interaction of the BepA edge-strand with the LptD β7 strand, we conducted site-specific disulfide crosslinking experiments. For this analysis, we used the above-described single Cys derivatives of BepA harboring a Cys residue at the position of Asn-105, Ala-106, or Phe-107, and derivatives of LptD having a Cys substitution at either of the six positions, including Tyr-331 at which introduction of pBPA showed clear crosslinking with BepA (Figure 3A). The wild-type LptD protein harbors intrinsic four Cys residues that form two disulfide bonds essential for the LptD function; therefore, each of these Cys-substituted LptD mutants possessed five Cys residues in total. We confirmed that these Cys-substituted LptD derivatives accumulated normally and retained their function (Figure 3—figure supplement 2). Cells expressing a combination of BepA Cys mutants and LptD Cys mutants were grown, and total proteins were acid-denatured and dissolved in SDS containing N-ethylmaleimide (NEM; NEM was included to block free Cys residues). Then, LptD-His10 and its crosslinked products were affinity-isolated using the C-terminal His10-tag, treated with or without 2-mercaptoethanol (ME), and analyzed by SDS-PAGE and anti-BepA immunoblotting. We observed that certain combinations of BepA and LptD derivatives showed a high-molecular-mass band in electrophoresis results. Among them, the combination of BepA(N105C) and LptD(Y331C) showed the most intense band that exhibited reaction with the anti-BepA antibody (Figure 3B and Figure 3—figure supplement 2B, no ME). These high-molecular-mass bands were not observed with the wild-type LptD (no additional Cys) and disappeared upon treatment with ME, suggesting that they were disulfide-crosslinked products (Figure 3B and Figure 3—figure supplement 2B, + ME). These results are consistent with the photo-crosslinking experiments (Figure 3A) and indicate that the edge-strand of BepA can directly bind to several regions in the N-terminal half of the LptD β-barrel-forming domain, which includes the β7 strand containing Tyr-331.

BepA interacts with an LptD intermediate associating with the seam region of BamA on the BAM complex

We further investigated the mode of the interaction of the BepA-associated LptD with the BAM complex. It has been recently shown that LptD4213, a mutant form of LptD that has a short (23 amino acids) deletion in an extracellular loop (eL4) and is stalled on the BAM complex mimicking a late assembly complex (Lee et al., 2016), interacts with the seam region formed by the N- and C-terminal β-strands (β1 and β16, respectively) in the BamA barrel domain and forms a hetero-complex, in which the C-terminal β-signal of the LptD4213 was associated with the β1 strand of the BamA seam (Lee et al., 2019). It has been suggested that the interaction of the β-signal with the BamA β1 strand generally facilitates the folding of the β-barrel domain of a substrate OMP (see Figure 4D and 5B; Tomasek and Kahne, 2021). We first examined the interaction of the BepA-associated LptD intermediate with BamA and BamD by conducting in vivo photo-crosslinking experiments using the LptD derivatives with pBPA at the position of Glu-749 or Tyr-726 in addition to the position of Tyr-331. pBPA at Glu-749 and Tyr-726 residue, both of which are located near the β-signal of LptD, have been reported to be crosslinked with BamA and BamD, respectively, during the LptD assembly (Figure 4D; Lee et al., 2019; Lee et al., 2018). Complementation assay results showed that either of the LptD derivatives containing one or two pBPA at Glu-749 and Tyr-726 were functional (Figure 4—figure supplement 1). After UV-irradiation of the cells expressing LptD-His10 and the pBPA derivatives, LptD-His10 and its crosslinked products were affinity-isolated from the membrane fractions and analyzed by immunoblotting. The single pBPA derivatives indeed generated crosslinked products with the expected factors as: LptD(Y331pBPA) with BepA, LptD(D749pBPA) with BamA, and LptD(Y726pBPA) with BamD (Figure 4A and B). With the double pBPA derivatives LptD(Y331/D749pBPA) and LptD(Y331/Y726pBPA), new crosslinked products with higher molecular sizes were observed in addition to the ones observed with single pBPA mutants (Figure 4A and B). These results showed that the higher molecular-sized products represented BepA–LptD(Y331/D749pBPA)–BamA and BepA–LptD(Y331/Y726pBPA)–BamD crosslinked products and that BepA interacts with an assembly intermediate of LptD on the BAM complex.

Figure 4 with 3 supplements see all
BepA interacts with an LptD intermediate assembling on the BAM complex.

(A, B) In vivo photo-crosslinking of an LptD mutant having pBPA at two positions with BepA. Cells of RM2243 (bepA(E137Q)) carrying pEVOL-pBpF, pMW118‐bepA(E137Q) and pRM294-lptD(amb)-his10 plasmids were grown at 30°C in L-medium containing 0.5 mM pBPA until early log phase and induced with 1 mM IPTG for 3 hr to express the indicated LptD(pBPA) variants. The cultures were divided into two portions, each of which was treated with or without UV-irradiation for 30 min at 4°C. Proteins of the total membrane fractions were subjected to pull-down with Ni-NTA agarose. Purified proteins were analyzed by 7.5% Laemmli SDS‐PAGE by immunoblotting with the indicated antibodies. Asterisk in the anti-BepA blots possibly indicates an LptD-BamA crosslinked product that was detected due to the apparent cross-reactivity of the anti-BepA antibody with the LptDxBamA crosslinked product (see below). (C) Simultaneous crosslinking of LptD having Y331pBPA and E733C with the BepA edge-strand and the seam region of BamA(S439C). Cells of RM3655 (bamA(S439C), ΔbepA)/pEVOL-pBpF/pMW118‐bepA(E137Q) carrying pRM294-lptD(E733C)-his10, or pRM294-lptD(Y331amb, E733C)-his10 were grown and induced as in (A). After treatment with or without BMB and the following quenching of BMB by addition of excess cysteine, the cultures were divided into two portions, each of which was treated with or without UV-irradiation for 30 min at 4°C. Total cellular proteins were acid-precipitated, solubilized with SDS buffer containing NEM, and subjected to pull-down with Ni-NTA agarose. Purified proteins were analyzed by 7.5% Laemmli SDS‐PAGE and immunoblotting with the indicated antibodies. The anti-BamA immunoblotting showed that the amount of the BepAxLptDxBamA crosslinked product was much lower than that of the LptDxBamA crosslinked product. Although the anti-BepA antibodies apparently cross-reacted weakly with the LptDxBamA crosslinked products (closed circles), the higher signal intensity of the BepAxLptDxBamA crosslinked product band as compared with the intensity of the LptDxBamA band (closed circles) indicate that the detection of the former band with the anti-BepA antibodies cannot be ascribed to this cross-reactivity. The identities of the bands marked by open circles in (A, B) are unclear; they might represent BepA-LptD crosslinked products or BepA-BamA crosslinked products (detected due to the cross-reactivity of anti-BepA antibodies with LptDxBamA crosslinked products as described above). In (A–C), we confirmed that the amounts of the isolated non-crosslinked LptD-His10 derivatives were roughly equal by CBB staining or anti-His immunoblotting (Figure 4—figure supplement 3). The result shown is a representative of two technical replicates. (D) A schematic cartoon of the interaction of the LptD assembly intermediate with BepA and BamA/D on the BAM complex. See Figure 4—source data 1 for gel images for (A–C).

Figure 4—source data 1

A Zip file containing gel images (A–C) for the immunoblotting experiments using the anti-BepA, anti-BamA, and anti-BamD antibodies.

https://cdn.elifesciences.org/articles/70541/elife-70541-fig4-data1-v2.zip
Figure 5 with 1 supplement see all
Model for the substrate recognition and discrimination by BepA.

(A) A schematic cartoon of the substrate recognition by BepA at its active site. See the text for details. (B) An overview of the proposed LptD assembly process and BepA-mediated discrimination of the assembling and stalled LptD species. See the text for details. Association of BepA with the assembly intermediate form of LptD on the BAM complex could transiently stabilize the LptD assembly intermediate and facilitate the association of LptE with LptD.

We further conducted photo-crosslinking experiments using LptD locked on the BAM complex by using an SH-crosslinker, 1,4-bismaleimidobutane (BMB). It has been previously shown (Lee et al., 2019) that a cysteine placed near the N-terminal β-strand of the BamA β-barrel (S439C) was crosslinked with a cysteine introduced near the β-signal of LptD (E733C) via BMB treatment (Figure 4D; Figure 4—figure supplement 2A). We introduced a Y331pBPA mutation into LptD(E733C) and confirmed that the resultant mutant was functional (Figure 4—figure supplement 2B). When the LptD(Y331pBPA/E733C) mutant was expressed in a strain having a chromosomal bamA(S439C) mutant gene, LptD–BepA crosslinked products were detected upon UV-irradiation, whereas LptD–BamA crosslinked products was detected upon BMB treatment. When cells were first treated with BMB and then UV-irradiated, a higher mass product that reacted with both anti-BepA and anti-BamA antibodies was generated. The generation of this product depended on both BMB-treatment and UV-irradiation (Figure 4C). These results are fully consistent with the above photo-crosslinking results and further demonstrated that BepA could interact with an LptD assembly intermediate associating with the seam site of BamA on the BAM complex.

Discussion

The involvement of BepA in the maintenance of structural and functional integrity of the OM was first suggested on the basis of increased sensitivities of the ΔbepA(yfgC) strain to several antibiotics and chemicals, which was similar to the characteristics of strains with a disruption of genes encoding proteins engaged in outer membrane biogenesis (Narita et al., 2013; Ruiz et al., 2005; Tamae et al., 2008). We have previously shown that BepA is involved in the biogenesis and quality control of LptD, probably on the BAM complex (Daimon et al., 2020; Daimon et al., 2017; Narita et al., 2013). However, the mechanism by which BepA recognizes and interacts with LptD remains elusive. This information is important to understand the mechanism of BepA to distinguish between the normal (on-) and off-pathway intermediates of LptD that are either assembled into the OM or degraded, respectively.

To gain insight into the BepA–LptD interaction, we examined the role of the conserved edge-strand of BepA in its function. Our results showed that the BepA edge-strand participates not only in the proteolytic activity but also, unexpectedly, in the chaperone-like activity through its direct interaction with LptD (Figure 1). The results of the photo- and disulfide-crosslinking analyses indicated that the N-terminal half of the LptD β-barrel-forming domain interacts with the BepA edge-strand (Figure 2 and Figure 3). Moreover, we showed that BepA demonstrated interaction with an LptD assembly intermediate whose C-terminal region was associated with the seam strand of BamA on the BAM complex (Figure 4). A ternary complex formation among an assembly intermediate of an OM protein (EspP, an autotransporter), a periplasmic chaperone (SurA/Skp), and the BAM complex has been suggested form biochemical studies including crosslinking, although the ternary complex was not directly detected (Ieva et al., 2011). We here experimentally demonstrated the ternary complex formation for LptD, BepA, and the BAM complex. A similar client-chaperone-BAM ternary complex might be formed in the assembly of other OM proteins. These observations provide useful insights into the BepA functions involved in the biogenesis and quality control of LptD, and also the assembly mechanism of other OM proteins.

Interaction between the edge-strand of BepA and an LptD assembly intermediate

Our previous results have shown that BepA interacts with the BAM complex via its C-terminal TPR domain partly inserted into the periplasmic ring-like structure of the BAM complex (Daimon et al., 2017; Shahrizal et al., 2019). In this study, we observed that BepA showed crosslinking with the N-terminal half of the LptD β-barrel-forming domain but not with the C-terminal half. This observation was consistent with the localization of LptD on the BAM complex; the N-terminal region was localized at/near the periplasmic surface of the BAM complex, and the C-terminal region was inserted deep into the BAM complex’s interior. The results of the disulfide-crosslinking experiments showed that several positions, including Tyr-331, in the N-terminal half of the LptD β-barrel-forming domain interacted with the edge-strand. The recently solved structures of BepA (Bryant et al., 2020; Shahrizal et al., 2019) showed that the active site region including the edge-strand was located inside the BepA molecule, leading to the suggestion that structural changes in BepA including the movement of the α6- and α9-loops covering the active site/edge-strand were necessary to enable access of a substrate to the active site/edge-strand region (Figure 5—figure supplement 1). However, even after such structural changes, the edge-strand should be located at a recessed position. This suggests that the regions around the BepA-crosslinked positions in the LptD assembly intermediate do not form an extensive β-sheet structure as found in the mature LptD to gain access to the edge-strand of BepA (Figure 5—figure supplement 1). Furthermore, pBPA at positions of both the inward-pointing and outward-pointing residues in the mature β-barrel domain of LptD was crosslinked with BepA.

Collectively, these results support the hypothesis that the BepA-interacting region of the LptD intermediate is largely unstructured. The unstructured nature of these proteins is in fact helpful to accommodate in or around the narrow space inside the BAM complex. The TPR domain of BepA has also been shown to contact with LptD to promote its biogenesis and degradation (Daimon et al., 2017). Currently, we have no information available on the part of LptD that interacts with the TPR domain. The TPR domain may act either together with the edge-strand at the same step or independently at other steps during LptD assembly and degradation.

The edge-strand and His switch mediate functional regulation of BepA in the assembly promotion and proteolytic quality control of LptD

Further, our results suggest that the proper interaction of LptD with the edge-strand of BepA is important for the promotion of its assembly as well as degradation by BepA (Figure 1 and Figure 5A). This finding was unexpected as it raised a question of how the 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 (Bryant et al., 2020; Shahrizal et al., 2019). We have recently reported that His-246 acts as a switch to regulate the proteolytic activity of BepA (Daimon et al., 2020). This His-switch-mediated repression of the protease activity would enable the interaction of LptD intermediates at the protease active site of BepA without degradation. Indeed, the derepressed BepA(H246A) mutant degrades an LptD intermediate on the normal assembly pathway, instead of promoting its biogenesis (Daimon et al., 2020).

While the exact role of the BepA's edge-strand in the chaperone-like activity remains unclear, our results raise the possibility that the edge-strand-mediated binding of a substrate is involved in the discrimination of LptD intermediates by BepA for assembly or degradation. One possible model would be as follows (Figure 5A). In both assembly and degradation pathways of LptD, an unknown signal(s), such as a specific interaction of BepA with the substrate and/or the BAM complex, induces a structural change in BepA (dislocation of the α6-loop covering the active site) to expose its active site and to enable the interaction between the LptD polypeptide and the edge-strand, whereas His-246 continues to repress the degradation of LptD by BepA. This interaction is necessary for both proper assembly and degradation of LptD. The transient interaction of normally assembling LptD with the ‘protease activity-repressed’ state of BepA would provide sufficient time for the progression of the maturation processes of LptD (including association with LptE), resulting in the final release of mature LptD from BamA. In contrast, during the prolonged stay of stalled or misassembled LptD molecules, BepA undergoes a further structural change including the movement of the α9-loop, stochastically or induced by a specific signal(s) that was transmitted from the BAM complex and/or LptD through their interaction with BepA, to remove His-246 from the active site zinc, leading to the degradation of the molecules that cannot be subjected to maturation successfully. Our previous results of pulse-chase experiments showed that, although the conversion of LptDC to LptDNC was relatively slow, it started immediately after the synthesis of LptD and proceeded rather constantly (Narita et al., 2013). In contrast, degradation of stalled LptD in an LptE-depleted strain apparently occurred in a biphasic manner; little degradation of stalled LptD was observed for several minutes after synthesis (Narita et al., 2013), indicating the presence of a lag period for degradation of stalled LptD to occur. These observations are consistent with the above model.

Notably, it has been reported that BepA expression can result in the degradation of a mutant form of LptD (LptD4213) that probably mimics a late-state assembly intermediate of LptD (Lee et al., 2019; Lee et al., 2016) that possesses a substantial degree of a higher order (β-barrel-like) structure and interacts with the Bam components (BamA and BamD), and LptE. The mechanism by which LptD4213 is degraded by BepA remains unclear, but its N-terminal region may interact with BepA.

A proposed function of BepA in the promotion of the assembly of an immature LptD and LptE

BepA possibly facilitates the association of LptD with LptE on the BAM complex as the phenotypes caused by the disruption of BepA, including drug sensitivity and retarded disulfide rearrangement in LptD, can be suppressed by the overproduction of LptE (Narita et al., 2013). We have previously detected two species of LptDC; one is not associated with LptE and observed only in an extremely early phase of its membrane assembly, and the other is associated with LptE and formed at a later phase (Miyazaki et al., 2018). It is likely that the LptDC molecule that was simultaneously crosslinked with BepA and BamA was not associated with LptE, as it was accumulated in the LptE-limiting condition; therefore, it would represent the former species of LptDC mentioned above. We assume that this LptD intermediate may possess a partially folded structure in which the BamA-associating C-terminal region of the barrel domain has a certain degree of a higher order (β-sheet) structure, but the BepA-associating N-terminal region is largely unstructured. Such a state of LptD may be favorable for the association of LptE with LptD (Figure 4D).

Interestingly, Tyr-331 of LptD that contacts with the edge-strand of BepA is located at the end of the LptE-surrounding region of the LptD β-barrel in the mature LptD/E complex (Figure 2D). The interaction with the edge-strand of BepA may pin the partially folded structure of LptD transiently and facilitate the association between LptD and LptE. It might also assist the formation of the β-sheet structure in the β-barrel-forming domain of LptD. Although it is possible that BepA actively promotes the LptD–LptE association by interacting with both LptD and LptE, it is also possible that BepA plays a passive role by maintaining an appropriate structure of LptD for association with LptE without showing direct interaction with LptE.

A model of the BepA-assisted biogenesis process of LptD

The biogenesis of LptD has been well studied. LptD has been a focus of OM protein research because it provides important information on its essential cellular function and it can be used as a model for OM protein insertion into the OM by the BAM complex. Based on the results obtained in the previous and current studies, we propose a model of the BepA-assisted biogenesis of LptD (Figure 5B): (i) After synthesis in the cytoplasm, LptD is translocated to the periplasm through the SecYEG translocon, during or just after which the Cys residues are oxidized by DsbA to form LptDC (Chng et al., 2012; Narita et al., 2013).

It is then targeted to the BAM complex with the aid of periplasmic chaperones including SurA (Schwalm et al., 2013; Vertommen et al., 2009). (ii) The C-terminal β-signal region of LptD is inserted into the BAM complex possibly through the periplasmic ring-like structure formed by the BamA POTRA domains and the lipoprotein subunits of the BAM complex, and it establishes interaction with BamD and BamA (Lee et al., 2019). (iii) Although details of the exact juncture at which interaction occurs are unknown, BepA with its protease activity repressed by His-246 residue interacts with the largely unstructured N-terminal half of the LptD β-barrel-forming domain whose C-terminal β-signal region is associated with the seam region of BamA. This stabilizes the partially unfolded assembly intermediate of LptD on the BAM complex to help the association of LptE with LptD. (iv) Then, the folding (β-sheet formation) of the unstructured N-terminal region of the LptD β-barrel domain occurs to form a premature form of LptD with a substantially folded β-barrel domain and LptE within it, like LptD4213 (Lee et al., 2019; Lee et al., 2016). (v) The β-barrel domain of LptD β-barrel is finally released from BamA and closed to form the mature LptD/E complex.

The isomerization of two disulfide bonds in LptD (LptDC to LptDNC) should occur at a later step after the association of LptD and LptE (Lee et al., 2016; Miyazaki et al., 2018). The LptD intermediates that are stalled at certain steps in the above-mentioned processes as a result of misfolding are eliminated by the action of several peptidases including DegP (in the periplasm) and BepA /YcaL (on the BAM complex) (Soltes et al., 2017).

Future perspectives of BepA study

Our study reports several new findings on the interaction of BepA with LptD and BamA on the BAM complex where BepA plays a crucial role in the biogenesis and degradation of LptD. Further, we proposed a model explaining the role of BepA in these processes. Nonetheless, there are many questions that warrant further investigation. It would be especially important to elucidate the mechanism by which the substrate gains access to the active site buried inside the BepA molecule and the manner in which the switching of BepA from the state with chaperone-like function to that with protease function occurs. The possible movement of the α6- and α9-loops should be directly examined. It is possible that there are signals that arise from the BAM complex and/or from (stalled) LptD to induce this structural/functional conversion of BepA. Further study, including structural and biochemical analysis of the BepA–LptD–BAM complex, is necessary to substantiate our model and to elucidate the molecular details of BepA functions. It remains an open question whether BepA also acts in the assembly and quality control of some other OM proteins, like in the case of LptD. Systematic identification and analysis of the additional BepA substrate OM proteins will be needed to know how generally BepA acts in biogenesis/degradation of OM proteins. Cell surface proteins are suitable drug targets as they are more easily accessible from the external milieu than cytoplasmic proteins. These studies can provide a basis for the development of new drugs targeted to BepA/LptD/BAM.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
strain, strain background (Escherichia coli)E. coli strainsThis studyN/ASupplementary file 1
strain, strain background (P1 bacteriophage)P1virLaboratory stockCGSC12133
recombinant DNA reagentPlasmidsThis studyN/ASupplementary file 2
sequence-based reagentPCR primersThis studyN/Adescribed in the below
antibodyPenta-His HRP conjugate(mouse monoclonal)QIAGEN34460(1:2000 or 1:3000 dilution)
antibodyAnti-BepA (rabbit polyclonal)Narita et al., 2013N/A(1:10000 dilution)
antibodyAnti-LptD (rabbit polyclonal)Narita et al., 2013N/A(1:50000 dilution)
antibodyAnti-BamA (rabbit polyclonal)Gunasinghe et al., 2018N/A(1:20000 dilution)
antibodyAnti-BamD (rabbit polyclonal)Gunasinghe et al., 2018N/A(1:10000 dilution)
antibodyGoat Anti-Rabbit IgG (H + L)-HRP ConjugateBio-Rad Laboratories1706515
RRID:AB_2617112
(1:5000)
chemical compound, drugH-p-Bz-Phe-OHBachemF2800
chemical compound, drugMethionine, L-[35S] Translation GradeAmerican Radiolabeled ChemicalsARS 01014
chemical compound, drugnProtein A Sepharose 4 Fast FlowGE Healthcare17528004
chemical compound, drugNi-NTA AgaroseQIAGEN30250
commercial assay or kitECL Western Blotting Detection ReagentsGE HealthcareRPN2106
commercial assay or kitECL Prime Western Blotting Detection ReagentsGE HealthcareRPN2232
software, algorithmMicrosoft ExcelMicrosoftRRID:SCR_016137
software, algorithmBio-imaging Analyzer BAS-1800, BAS-5000Fujifilm/GE HealthcareN/A
software, algorithmImage Qaunt LAS 4000 miniFujifilm/GE HealthcareN/A
software, algorithmMulti GaugeFujifilm/GE HealthcareRRID:SCR_014299

Bacterial strains and plasmids

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E. coli K12 strains and plasmids used in this study are listed in Supplementary files 1 and 2, respectively. Details of the strain and plasmid construction and media are described in Construction of mutant strains and Plasmids construction, respectively.

Media and bacterial cultures

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E. coli cells were grown in L-rich medium (10 g/L bacto-tryptone, 5 g/L bacto-yeast extract, 5 g/L NaCl; pH adjusted to 7.2 with NaOH) or M9 synthetic medium (without CaCl2; Miller, 1972) supplemented with maltose (final 0.2%), glycerol (final 0.4%), all amino acids (except Met and Cys; final concentration of 20 μg/mL each). 50 µg/mL ampicillin (Amp), 20 µg/mL chloramphenicol (Cm), 25 µg/mL kanamycin (Km), 25 µg/mL tetracycline (Tet), and 50 µg/mL spectinomycin (Spc) were added as appropriate for growing plasmid-bearing cells and selection of transformants and transductants. Bacterial growth was monitored with Mini photo 518R (660 nm; TAITEC Co., Saitama, Japan).

Construction of mutant strains

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RM2091 (JE6631, purC80::Tn10) was constructed by transferring the purC80::Tn10 marker, which is located near the bepA gene, from CAG18470 (Nichols et al., 1998) into JE6631 (Miyazaki et al., 2018), respectively, by P1 transduction. RM2243 (AD16, bepA(E137Q) purC80::Tn10) was constructed as follows. pRM330 (a plasmid carrying bepA(E137Q), see below) was introduced into RM2091 to yield cells with pRM330 integrated into the chromosome by homologous recombination in the bepA region. They were then grown on an L-agar plate containing 5% sucrose to select cells that had lost the integrated plasmid. The plasmid-cured cells were screened for those having the chromosomal bepA(E137Q) allele at the bepA locus. The bepA(E137Q) allele was finally transferred to AD16 (Kihara et al., 1995) by joint P1 transduction with the purC80::Tn10 marker. One of such strains was named RM2243. RM3654 (AD16, ΔbepA, bamA+ zae502::Tn10) and RM3655 (AD16, bamA(S439C) zae502::Tn10) were constructed as follows, pRM845 (a plasmid carrying bamA(S439C), see below) was introduced into YH188 (JE6631, zae502::Tn10) (Hizukuri and Akiyama, 2012) to yield cells with pRM845 integrated into the chromosome by homologous recombination in the bamA region. They were then grown on an L-agar plate containing 5% sucrose to select cells that had lost the integrated plasmid. The plasmid-cured cells were screened for those having the chromosomal bamA(S439C) allele at the bamA locus. The bamA(S439C) allele was transferred to SN56 (Narita et al., 2013) by joint P1 transduction with the zae502::Tn10 marker. Strains having the bamA+ allele and the bamA(S439C) allele were picked up and named RM3654 and RM3655, respectively. RM2831 (HM1742, kan araC-ParaBAD-lptD) were constructed by essentially the same procedure as the construction of strains with a chromosomal C-terminal his10-tagged gene (Miyazaki et al., 2020b). First, a kan araC-ParaBAD-lptD fragment having a sequence identical to the upstream or downstream region of the lptD start codon at the respective ends of the fragment, was PCR-amplified from pRM741 (a plasmid carrying a kan cassette at the upstream of an araC-ParaBAD) using a pair of primers, ara-lptD-f (5′-TTGTCACGCGCAACGTTACCGATGATGGAACAATAAAATCAACGTCATATGAATATCCTCCTTAG-3′) and ara-lptD-r (5′-GGTGGCAATCATGGTGGCCAGGAGAGTGGGGATACGTTTTTTCATGGTGAATTCCTCCTGCTAG-3′). Then, the chromosomal lptD locus of the E. coli DY330 strain was replaced by this fragment using the λ-Red recombination system (Yu et al., 2000). The kan araC-ParaBAD-lptD was finally transferred to HM1742 by P1 transduction.

Plasmids construction

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pSTD689-derived plasmids carrying a bepA mutants were constructed from pRM290 (pSTD689-bepA) (Daimon et al., 2017) by site-directed mutagenesis. Derivatives of pRM291 (pSTD689-bepA(E137Q)) (Daimon et al., 2017) carrying an additional Cys mutation and derivatives of pUC-bepA(E137Q)-his10 (pUC18-bepA(E137Q)-his10) (Narita et al., 2013) carrying an amber mutation were constructed by site-directed mutagenesis. pUC18-bepA(Pro)-his10 and pUC18-bepA(amb)-his10 plasmids were constructed from pUC-bepA-his10 (pUC18-bepA-his10) (Narita et al., 2013) by site-directed mutagenesis. To construct pUC18-bepA(Pro, amb, E137Q)-his10 plasmids, a mutation for the individual Pro substitutions was introduced into each of the pUC18-bepA(amb, E137Q)-his10 plasmids by site-directed mutagenesis. pNB91 (pMW118-bepA(E137Q)) was constructed by subcloning an EcoRI-HindIII bepA(E137Q) fragment prepared from pUC-bepA(E137Q) (pUC18-bepA(E137Q)) (Narita et al., 2013) into the same sites of pMW118. pRM807 (pMW118-bepA(A106P, E137Q)) and pRM808 (pMW118-bepA(F107P, E137Q)) were also constructed by subcloning an EcoRI-HindIII bepA fragment from each of pSTD639-bepA plasmids into the same sites of pMW118.

Plasmids carrying lptD(amb)-his10 were constructed from pRM309 (pRM294-lptD-his10) (Miyazaki et al., 2018) by site-directed mutagenesis. pRM821 (pRM294-lptD(Y331amb, D749amb)-his10) and pRM822 (pRM294-lptD(Y331amb, Y726amb)-his10) were constructed from pRM626 (pRM294-lptD(Y331amb)-his10) by site-directed mutagenesis. pRM829 (pRM294-lptD(E733C)-his10) and pRM831 (pRM294-lptD(Y331, E733C)-his10) were constructed from pRM309 and pRM626, respectively, by site-directed mutagenesis. Plasmid carrying lptD(Cys)-his10 were constructed as follows. Each mutation for the Cys substitutions was introduced to pRM309 by site-directed mutagenesis. The BamHI-HindIII lptD(Cys)-his10 fragment of the resulting plasmids was subcloned into the same site of pTWV228 (Takara Bio Inc, Shiga, Japan) to generate pTWV228- lptD(Cys)-his10.

pRM320 (pUC118-bepA-yfgD) was constructed by PCR amplification of the bepA-yfgD fragment from the genome of MC4100 using a pair of primers, bepA-f (5′-GCGCGCGGATCCATTTGAGTGGGCTAATCTTCG-3′) and yfgD-r (5′-GCGCGCGTCGACCGAACTACGCGAAGTTAATCC-3′), and, cloning it into the BamHI-SalI site of pUC118 (Takara Bio Inc) after digestion with these enzymes. For the construction of pRM324, the bepA(E137Q) mutation was introduced into pRM320 by site-directed mutagenesis. pRM330 (pK18mobsacB-bepA(E137Q)-yfgD) was constructed by subcloning the BamHI-SalI bepA(E137Q)-yfgD fragment from pRM324 into the same sites of pK18mobsacB (Schäfer et al., 1994).

pRM823 (pUC118-bamA) was constructed by in vitro recombination using In-Fusion HD Cloning Kit (Takara Bio Inc) of an EcoRI-BamHI fragment from pUC118 and a bamA fragment prepared by PCR amplification from the genome of MC4100 using a pair of primers, bamA-f (5′-GCGCGAATTCAGGAAGAACGCATAATAACG-3′) and bamA-r (5′-GCGCGGATCCTTACCAGGTTTTACCGATG-3′). For the construction of pRM836, the bamA(S439C) mutation was introduced into pRM823 by site-directed mutagenesis. pRM845 (pK18mobsacB-bamA(S439C)) was constructed by subcloning the EcoRI-BamHI bamA(S439C) fragment from pRM823 into the same sites of pK18mobsacB.

Immunoblotting analysis

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Acid-denatured proteins were solubilized in SDS-sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5 mg/mL bromophenol blue) with or without 10% β-ME, boiled at 98°C for 5 min, separated by SDS-PAGE and electro-blotted onto a PVDF membrane (Merck Millipore; Billerica, MA). The membrane was first blocked with 5% skim milk in PBST (Phosphate Buffered Saline with Tween 20), and then incubated with Penta-His HRP conjugate (1:2000 or 1:3000 dilution), anti-BepA (1:10,000), anti-LptD (1:50,000), anti-BamA (1:20,000), or anti-BamD (1:10,000). After washing with PBST, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) (Goat Anti-Rabbit IgG (H+L)-HRP Conjugate; Bio-Rad Laboratories, Inc, Hercules, CA) in PBST (this step was omitted for the detection using Penta-His HRP Conjugate). Proteins were visualized with ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd, Amersham Place Little Chalfont, England) or ECL Prime Western Blotting Detection Reagents (GE Healthcare) and LAS4000 mini lumino-image analyzer (GE Healthcare).

Pulse-chase analysis for assay of the LptD disulfide-isomerization

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Cells were first grown at 30°C in M9-medium supplemented with 2 µg/mL thiamine, 0.4% glycerol, 0.2% maltose, all amino acids (except Met and Cys) with or without 0.05% arabinose until early log phase. After induction with 1 mM IPTG for 15 min, cells were pulse-labeled with 370 kBq/mL [35S]methionine for 1 min. At appropriate time points after addition of excess nonradioactive Met (final conc. 250 µg/mL), total cellular proteins were precipitated with 5% TCA, washed with acetone, solubilized in SDS buffer (50 mM Tris-HCl (pH 8.1), 1% SDS, and 1 mM EDTA) and diluted 33-fold with Triton buffer (50 mM Tris-HCl (pH 8.1), 150 mM NaCl, 2% Triton X-100, and 0.1 mM EDTA). After clarification, samples were incubated with anti-LptD antibodies and nProtein A Sepharose 4 Fast Flow (GE Healthcare) at 4°C overnight with slow rotation. Proteins bound to the antibody/ProteinA-Sepharose were recovered by centrifugation, washed with Triton buffer, and then with 10 mM Tris-HCl (pH 8.1) and eluted by incubation at 98°C for more than 5 min in SDS-sample buffer. The samples were divided into two portions and one was treated with 10% β-ME. The proteins were separated by SDS-PAGE, and visualized with BAS1800 phosphoimager. Relative LptDNC were calculated by the following equation: Relative LptDNC=[LptDNC(X min)]/[LptDC(5 min)+LptDNC(5 min)], where LptDNC and LptDC are the intensities of the respective bands.

In vivo photo-crosslinking analysis, pEVOL-pBpF expresses an evolved tRNA/aminoacyl-tRNA synthetase pair that enable in vivo incorporation of pBPA into an amber codon site of a target protein by amber suppression. UV exposure of a cell expressing a pBPA-incorporated protein causes the generation of a covalent crosslinking between the pBPA in the target protein and a nearby protein, allowing the detection of their in vivo interaction (Chin and Schultz, 2002; Miyazaki et al., 2020a; Young et al., 2010). For the experiments in Figure 1D and Figure 3—figure supplement 1, cells were grown at 30°C in L-medium containing 0.5 mM pBPA and 0.02% arabinose until early log phase and induced with 1 mM IPTG for 1 hr. A half volume of the cell cultures was put on a petri dish and UV-irradiated at 4°C for 10 min using B-100AP UV lamp (365 nm; UVP, LLC, Upland, CA), at the distance of 4 cm. The other half was kept on ice as non-UV-irradiated samples. Membrane fractions were prepared by sonical cell disruption and the following ultracentrifugation, and solubilized with SDS buffer (50 mM Tris-HCl (pH 8.1), 1% SDS, and 1 mM EDTA). After 33-fold diluted with Triton buffer (50 mM Tris-HCl (pH 8.1), 150 mM NaCl, 2% Triton X-100, and 0.1 mM EDTA) and clarification, samples were subjected to pull-down with Ni-NTA Agarose (QIAGEN). The isolated proteins were solubilized in SDS-sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5 mg/mL bromophenol blue) containing ME, boiled at 98°C for 5 min, and analyzed by SDS-PAGE and immunoblotting analysis.

For the experiments in Figures 2A, B and 3A, cells were grown at 30°C in L-medium containing 0.5 mM pBPA and 0.02% arabinose until early log phase and induced with 1 mM IPTG for 3 hr. While a half volume of the cell cultures was UV-irradiated for 10 min, the other half was kept on ice as non-UV-irradiated samples, as above. Total cellular proteins were precipitated with 5% TCA, washed with acetone, and solubilized in SDS-sample buffer containing ME and boiled at 98°C for 5 min. The samples were subjected to SDS-PAGE and immunoblotting analysis.

For experiments in Figure 4A and B, cells were grown at 30°C in L-medium containing 0.5 mM pBPA and 0.02% arabinose until early log phase and induced with 1 mM IPTG for 3 hr. While a half volume of the cell cultures was UV-irradiated for 30 min, the other half was kept on ice as non-UV-irradiated samples, as above. Membrane fractions were prepared as above and solubilized in SDS buffer. After dilution with Triton buffer and clarification, samples were subjected to pull-down with Ni-NTA Agarose. The isolated proteins were solubilized in SDS-sample buffer containing ME, boiled at 98°C for 5 min, and analyzed by SDS-PAGE and immunoblotting analysis.

BMB crosslinking combined with photo-crosslinking

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Cells were grown at 30°C in L-medium containing 0.5 mM pBPA and 0.02% arabinose until early log phase and induced with 1 mM IPTG for 3 hr. BMB crosslinking was performed essentially according to the previously described procedures (Lee et al., 2019). After quenching the BMB crosslinking, cells were treated with or without UV-irradiation for 30 min at 4°C. Total cellular proteins were precipitated with 5% TCA, washed with acetone, and suspended in SDS-sample buffer containing 12.5 mM NEM for blocking the free Cys residues. After dilution with Triton buffer and clarification, samples were subjected to pull-down with Ni-NTA Agarose. The isolated proteins were suspended in SDS-sample buffer containing ME, boiled at 98°C for 5 min, and analyzed by SDS-PAGE and immunoblotting analysis.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, and 4, Figure 1-figure supplements 2, 3, and 4, Figure 2-figure supplements 1, and 2, Figure 3-figure supplements 1, and 2, and Figure 4-figure supplements 1, 2, and 3.

References

  1. Book
    1. Miller JH
    (1972)
    Experiments in Molecular Genetics
    Cold Spring Harbor Laboratory Press.

Decision letter

  1. Gisela Storz
    Senior Editor; National Institute of Child Health and Human Development, United States
  2. Heedeok Hong
    Reviewing Editor; Michigan State University, United States
  3. Heedeok Hong
    Reviewer; Michigan State University, United States
  4. Yihua Huang
    Reviewer

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

This is a wonderful study reporting a critical role of the edge-strand of the periplasmic metalloprotease BepA in the equality control of an outer membrane protein in gram-negative bacteria. This result provides new insights into the biogenesis mechanisms of outer membrane proteins and the maintenance of outer membrane integrity.

Decision letter after peer review:

Thank you for submitting your article "Edge-strand of BepA interacts with immature LptD on the β-barrel assembly machine to direct it to on-and off-pathways" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, , including Heedeok Hong as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Gisela Storz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yihua Huang (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. Requested experiments: the following experiments will help strengthening the conclusions.

1a) This is a suggestion that can support authors' conclusion that BepA has a chaperone-like activity. Although authors showed that A106P retains proteolytic activity, a test seems needed for other mutations. The interpretation of Figure 1 data is based on the assumption that the mutations only locally perturb the structure of BepA not severely affecting the structural integrity of other regions. Would an in vitro proteolytic assay using a structureless substrate be possible at an elevated temperature?

1b) In Figure 4c and Figure 3B, a β-ME treatment control should be added in the supporting information to confirm the crosslinking band of BepA×LptD×BamA.

2. Clarification is needed whether the chaperone-like activity facilitates LptD maturation without proteolysis, or involves proteolysis as a part of quality control mechanisms. If the latter is true, can we still say that BepA is a chaperone? Also, please, add more explanations regarding the molecular mechanisms that underlie the chaperone-like activity.

2a) Line 151-Line 160 and Figure 1D: the evidence of the pulse-chase experiments to examine the chaperone-like function of BepA is relatively weak. Since the N105P, A106P and F107P mutants disrupt the interaction with LptD and they have weaker proteolytic activities, these could result in LptDC accumulation to make the radio of LptDNC lower for these mutants. It's difficult to ignore the effects of the proteolytic activities in this assay. Please, clarify this point.

2b) I recommend that authors add an additional sentence to lines 161-169, for example, "It is unclear at this point whether the apparent chaperone-like activity is due to proteolysis or not." or clarify this point in another way to guide readers.

2c) In Discussion, it will be helpful to describe a possible molecular principle of how the binding of the edge-strand to the substrate can lead to chaperone activity.

3. Please, emphasize broader implications of this work in OMP quality control in a way that this manuscript can better fit to the scope of eLife. Also, the arguments for the unique findings of this work in comparison to related studies need to be strengthened further.

3a) For example, a general reader would want to know more about:

3a-i) In what other biological processes is BepA involved?

3a-ii) Is LptD the only known substrate of BepA?

3a-iii) Is the involvement of the ternary complex (i.e., BAM, an OMP client, and a quality control enzyme) a novel finding in the biogenesis of OMPs? Also, can the proposed mechanism be generalizable to OMP quality control?

3b) Reviewer 3 raises a concern that the impact of this work is incremental rather than substantial in the field of OM/OMP biogenesis. Please, provide convincing arguments on the following points:

3b-i) It is possible that the edge strand may just be reporting on substrate binding to the protease domain active site. While this may be important for substrate recognition, it does not mean that the edge strand-substrate interaction plays a deterministic role in subsequent protein triage during LptD assembly. The role of the edge strand in substrate binding has been known for other M48 metalloprotease family proteins.

3b-ii) The manuscript describes in detail how BepA likely establishes the interaction with immature LptD on the Bam complex, but many of the general conclusions into the mechanism of LptD assembly have already been described elsewhere both by this group and others. As a result, the data in the work provides limited additional insight to the general mechanism of action of BepA.

3b-iii) Figure 4 and 5: The models presented in Figures 4D, 5A, and 5B are essentially reproductions from Lee 2019, Daimon 2020, and Tomasek 2020. The main conceptual advance to the model is largely explained by Figure 4D and I'm not sure Figure 5 is warranted given its limited additional insight. I would perhaps move the entirety of Figure to the figure-supplements.

Reviewer #1 (Recommendations for the authors):

1. This study is highly focused on BepA-LptD-BAM although the result can have a broad impact as a mode of protein quality control mechanisms in general. Readers may be lost in the midst of the detailed descriptions of experimental systems (M48 metalloproteases, BepA, LptD, LptD4213, etc.) and experimental results.

I recommend that authors emphasize broader implications of this work in the introduction and discussion. For example, readers would want to know more about:

Is LptD the only substrate of BepA?

What other biological processes is BepA involved?

Is the involvement of the ternary complex (i.e., BAM, a OMP client, and a quality control enzyme) in LptD biogenesis a novel finding in the field?

Would the proposed mechanism be generalizable to other BepA-mediated quality control of OMPs?

2. Discussion is excellent. Nonetheless, it could be more easily conveyed to readers if it is divided into several subsections with proper headings.

3. Clarification of the LptD mutant, LptD4213, is needed (in one sentence).

Reviewer #2 (Recommendations for the authors):

In all, this manuscript provides novel and detailed findings regarding the interaction of BepA with LptD and BamA in the BAM complex where BepA plays a crucial role in the biogenesis and degradation of LptD. The experiment designs are ingenious; the data presented in the manuscript are elegant and generally support the conclusions. I recommend this manuscript for publication if the following issues are currently addressed.

1. Line 151-Line 160 and Figure 1D, the evidence of the pulse-chase experiments to examine the chaperone-like function of BepA is relatively weak. Since the N105P, A106P and F107P mutants disrupt the interaction with LptD and they have weaker proteolytic activities, these could result in LptDC accumulation to make the radio of LptDNC lower for these mutants. It's difficult to ignore the effects of the proteolytic activities in this assay.

2. Does BepA only degrade LptDC not LptDNC?

3. In Figure 4c and Figure 3B, a β-ME treatment control should be added to further confirm the crosslinking band of BepA×LptD×BamA.

4. Line 161-Line 165, it is not very clear why the authors performed Cys substitution for positions of N-105, A106, or F107? If they try to draw the conclusion that "the secondary structure of the edge-strand, but not the individual amino acid residues, is important for its function", they may mutate the individual position to different types of residues for experiments.

5. For Figure 1, the authors should clearly label out the catalytic residues mentioned in the main text.

6. In general, it is a little bit hard to understand how come the edge-stand of BepA is able to directly contact with its substates as this this strand (β2) appears to be buried in the crystal structure of BepA. The authors should discuss this in the Discussion part.

7. The crosslinking assay revealed that BepA can interact with an LptD intermediate assembling on BAM complex. Does the dual function of BepA depend on the BAM complex?

Reviewer #3 (Recommendations for the authors):

My main criticism is the conclusion that the edge strand of BepA plays an active role to direct it (LptD) to on- and off- pathways. This is possible, but it would be important to show a mutation in the edge strand that could uncouple the chaperone and proteolytic activities of BepA. In both the A106P and F107P mutants, the loss in proteolytic activity is always accompanied by a loss in chaperone activity. I recognize that identifying such a mutant is not trivial, but the title of the paper implies that the edge strand plays a larger role in the functional switch in BepA.

As is, the manuscript describes in detail how BepA likely establishes the interaction with immature LptD on the Bam complex. but many of the general conclusions into the mechanism of LptD assembly have already been described elsewhere both by this group and others. As a result, the data in the work provides limited additional insight to the general mechanism of action of BepA.

Detailed changes:

1) I would consider including a reference and an explanation describing the antibiotic sensitivity and complementation assays. Specifically, I think it would be helpful to explain how you can use erythromycin sensitivity to test BepA function in the antibiotic sensitivity assays and to explain how glucose/arabinose/IPTG regulate protein levels in the strains used for the complementation assays. These details may not be obvious to everyone and would be helpful for a more general readership. The description can be short and described in the figure captions.

2) All the structure figures look a little fuzzy and should be rendered at a higher resolution. This may be an artifact of the compression during submission, though.

3) Figure 1A (right): The 'catalytic residues' label is misspelled. The green in Regulatory His246 is also hard to read. I recommend changing the color.

4) Figure 1D: I am weary about over interpreting the quantification of relative LptDNC levels, especially with the BepA F107P variant. I'm not sure if this is the best way to characterize BepA chaperone activity and the overall conclusions of this experiment still hold even if the quantification is removed.

5) Figure 2B and 3A: LptD pBPA E391 and Q393 both change the migration pattern of LptD rather noticeably. Do you know why this is the case? BPA substitution causes some changes at other positions, but these two in particular stand out. It is almost as if there is a deletion.

6) Figure 4: Figure 4C seems to suggest that the BepA antibody may also weakly cross react with BamA. If that's the case, then it is likely that the higher of the two molecular weight adducts labeled as BepAxLptD in both Figure 4A and 4B (lane 7) are actually LptDxBamA adducts. This band should be labeled with an asterisk instead.

7) Figure 4 and 5: The models presented in Figures 4D, 5A, and 5B are essentially reproductions from Lee 2019, Daimon 2020, and Tomasek 2020. The main conceptual advance to the model is largely explained by Figure 4D and I'm not sure Figure 5 is warranted given its limited additional insight. I would perhaps move the entirety of Figure to the figure-supplements.

https://doi.org/10.7554/eLife.70541.sa1

Author response

Essential revisions:

1. Requested experiments: the following experiments will help strengthening the conclusions.

1a) This is a suggestion that can support authors' conclusion that BepA has a chaperone-like activity. Although authors showed that A106P retains proteolytic activity, a test seems needed for other mutations. The interpretation of Figure 1 data is based on the assumption that the mutations only locally perturb the structure of BepA not severely affecting the structural integrity of other regions. Would an in vitro proteolytic assay using a structureless substrate be possible at an elevated temperature?

Thank you for the suggestion. Unfortunately, we have not succeeded in reproducibly detecting the proteolytic activity of BepA with purified BepA even when an unstructured substrate (a-casein) is used and the assay was conducted at an elevated temperature, possibly because the proteases activity of isolated BepA is tightly repressed by the mechanism that included His-246-mediated regulation as described in our paper (please see Introduction). Although BepA mutants with a mutation of His-246 or a deletion of H9 loop (these mutations release the His-246-mediated repression) significantly degrade a-casein, a combination of these mutations with the edge-strand mutations should make the interpretation of the results complicated. We thus think that the suggested experiments cannot be conducted soon.

Instead, we described the following points in the revised manuscript. Although we mentioned the self-cleavage activity of only the A106P mutant in the original manuscript, our results showed that the other edge-strand Pro mutants (other than F107P) exhibited significant self-cleavage activities as well (Figure 1—figure supplement 2B). In addition, the Pro mutants other than the A106P mutant degraded misfolded or unfolded BamA at a detectable level (Figure 1—figure supplement 2A). Furthermore, all the Pro mutants accumulated at a level comparable to that of wild-type BepA. These observations together indicate that the Pro mutations specifically affected the edge-strand structure, but not drastically altered the active site or the protein's overall structures. We described the above points in the revised text (line 174 in p7 to line 181 in p8). The differential effects of the Pro mutations on the degradation of LptD and BamA, and the self-cleavage of the C-terminal tag (Figure 1B and Figure 1—figure supplement 2A and 2B) might reflect their different interaction properties and/or affinities for the BepA edge-strand, as stated in lines 140–142 in p6.

1b) In Figure 4c and Figure 3B, a β-ME treatment control should be added in the supporting information to confirm the crosslinking band of BepA×LptD×BamA.

For Figure 4C, as the crosslinker used (BMB) is not cleavable with β-ME, we did not include a β-ME treatment control. However, the presumed crosslinked product between BamA(S430C) and LptD(E773C) was generated in a BMB- and Cys-dependent manner (Figure 4—figure supplement 2A, see below), strongly suggesting that it was indeed the BMB-mediated crosslinked product between BamA and LptD. For Figure 3B, a β-ME treatment control had been shown in the original figure (the middle panel). Please note that in this experiment, the crosslinked products were purified by making use of the His-tag attached to LptD, and detected by anti-BamA immunoblotting. Thus, no crosslinked product was detected after the β-ME treatment.

We noticed that the original Figure 4—figure supplement 2A contained several errors: aBepA should read aBamA, and LptD-His10xBepA should read LptD-His10xBamA. These errors have been corrected in new Figure 4—figure supplement 2A. We sincerely apologize to the editors and the reviewers for these errors.

2. Clarification is needed whether the chaperone-like activity facilitates LptD maturation without proteolysis, or involves proteolysis as a part of quality control mechanisms. If the latter is true, can we still say that BepA is a chaperone? Also, please, add more explanations regarding the molecular mechanisms that underlie the chaperone-like activity.

2a) Line 151-Line 160 and Figure 1D: the evidence of the pulse-chase experiments to examine the chaperone-like function of BepA is relatively weak. Since the N105P, A106P and F107P mutants disrupt the interaction with LptD and they have weaker proteolytic activities, these could result in LptDC accumulation to make the radio of LptDNC lower for these mutants. It's difficult to ignore the effects of the proteolytic activities in this assay. Please, clarify this point.

Thank you for pointing out the problem of the data presentation. We re-analyzed the pulse- chase data. To examine only the chaperone-like activities (promotion of the LptDNC generation) without the possible effect of the LptD degradation by the residual proteolytic activities of the A106P and the F107P mutants, the relative amounts of LptDNC at each chase point to the total LptD (LptDC + LptDNC) at the 5 min chase point were calculated and plotted as quantified data (new Figure 1D, right panel). The data clearly showed that the expression of WT BepA in the DbepA strain accelerated the maturation of LptD (that is, generation of LptDNC). Also, the A106P and the F107P mutant were mostly defective in the promotion of the LptD maturation. These results were described in the revised manuscript (lines 156–166 in p7). In addition, we previously showed that the expression of the protease-dead mutants, E137Q and H136R, significantly promoted the LptD maturation (please see Figure 4 in Narita et al. PNAS, 110, E3612–E3621, 2013; doi:10.1073/pnas.1312012110) and partially suppressed the OM-defective phenotypes of the bamB/bepA and bamE/bepA double-knockout strains (Figure 6 in Narita et al. PNAS, 110, E3612–E3621, 2013; doi:10.1073/pnas.1312012110) (note that in Figure 1D of the current work, the expression of the E137Q mutant only slightly promoted the LptD maturation. This would be ascribable to some difference in the experimental conditions including a lower expression level of the BepA mutant protein in the experiment for Figure 1D). These results collectively show that BepA possesses the chaperon-like activity (LptD maturation promoting activity) independent of its protease activity.

2b) I recommend that authors add an additional sentence to lines 161-169, for example, "It is unclear at this point whether the apparent chaperone-like activity is due to proteolysis or not." or clarify this point in another way to guide readers.

Thank you for the suggestion. However, as stated above, we think that our previous and current data indicated that BepA possesses the chaperone-like activity independent of its protease activity. We thus consider it inappropriate to add the suggested sentence. But, if you still think that the inclusion of the suggested sentence is essential, we are willing to do so.

2c) In Discussion, it will be helpful to describe a possible molecular principle of how the binding of the edge-strand to the substrate can lead to chaperone activity.

We suppose that the interaction of BepA (via the edge-strand) with an assembly intermediate of LptD on the BAM complex stabilizes the partially unfolded assembly intermediate of LptD on the BAM complex to help the association of LptE with LptD. Sentences explaining this point were added to Discussion (lines 388–392 in p16) and the legend to Figure 5.

3. Please, emphasize broader implications of this work in OMP quality control in a way that this manuscript can better fit to the scope of eLife. Also, the arguments for the unique findings of this work in comparison to related studies need to be strengthened further.

3a) For example, a general reader would want to know more about:

3a-i) In what other biological processes is BepA involved?

Our previous (Figure 8B in Daimon et al. Mol. Microbiol. 106, 760-776, 2017; DOI:10.1111/mmi.13844) and the current (Figure 1—figure supplement 2A in this paper) results showed that BepA can degrade misassembled (and possibly misfolded or unfolded) BamA in the DsurA strain. Thus, BepA might also act in the proteolytic quality control of other OMPs including BamA. It remains an open question whether BepA also acts in the assembly of some other OM proteins, like in the case of LptD. Systematic identification and analysis of the additional BepA substrates will be needed to know how generally BepA acts in the biogenesis/degradation of OM proteins.

These points were described in the revised text (lines 71–73 in p3 and lines 411–412 in p16).

3a-ii) Is LptD the only known substrate of BepA?

Please see our response to (3a-i).

3a-iii) Is the involvement of the ternary complex (i.e., BAM, an OMP client, and a quality control enzyme) a novel finding in the biogenesis of OMPs? Also, can the proposed mechanism be generalizable to OMP quality control?

A ternary complex formation among an assembly intermediate of an OM protein (EspP), a periplasmic chaperone (SurA/Skp), and the BAM complex has been suggested from biochemical studies including crosslinking, although the ternary complex was not directly detected (Ieva et al. PNAS, E383-391, 2011; doi:10.1073/pnas.1103827108). We here experimentally demonstrated the ternary complex formation for LptD, BepA, and the BAM complex. A similar client-chaperone-BAM ternary complex might be formed in the assembly of other OM proteins. This should be addressed in a future study.

The above points were described in the revised text (lines 292–297 in p12 and lines 409–412 in p16).

3b) Reviewer 3 raises a concern that the impact of this work is incremental rather than substantial in the field of OM/OMP biogenesis. Please, provide convincing arguments on the following points:

3b-i) It is possible that the edge strand may just be reporting on substrate binding to the protease domain active site. While this may be important for substrate recognition, it does not mean that the edge strand-substrate interaction plays a deterministic role in subsequent protein triage during LptD assembly. The role of the edge strand in substrate binding has been known for other M48 metalloprotease family proteins.

Our data demonstrated that the edge-strand of BepA directly binds a substrate. As pointed out by the reviewer, the involvement of the edge-strand in substrate binding has been known for other proteases. However, it was not known whether the substrate binding at the edge-strand contributes to the chaperone-like function; it was possible that the binding sites of a substrate on BepA during its proteolysis and its maturation are totally different as the chaperone-like activity of BepA is independent of its protease activity (it was conceivable, for example, that substrate binding during it maturation occurs on the surface of the C-terminal TPR domain that has been shown to interact with LptD). Our results showed that the defective binding of a substrate (LptD) at the edge-strand impairs not only its proteolysis but also its normal maturation (assembly). Because the edge-strand-bound substrate would be directly presented to the proteolytic active site for its degradation, this binding step should be important for the determination of the fates of the bound substrate. Our results strongly suggest that the substrate binding by the edge-strand is a crucial common step required for the subsequent protein triage during the LptD assembly.

3b-ii) The manuscript describes in detail how BepA likely establishes the interaction with immature LptD on the Bam complex, but many of the general conclusions into the mechanism of LptD assembly have already been described elsewhere both by this group and others. As a result, the data in the work provides limited additional insight to the general mechanism of action of BepA.

The results by us and others have previously shown that BepA has both proteolytic and chaperone-like (that is, LptD-assembly promoting) activities. Although it has been easily expected that the edge-strand-mediated interaction with a substrate is important for its degradation, it is not known how BepA executes its chaperone-like function and how BepA distinguishes the on- and off-pathway substrates. As described above, we showed that the normal interaction of the edge-strand of BepA with LptD is required not only for the degradation but also, unexpectedly, for the assembly promotion of this OMP substrate. Also, we demonstrated that BepA forms a ternary complex with LptD and the BAM complex in which BepA interacts with an unstructured N-terminal half of the LptD barrel domain and that the C-terminal region of LptD is associated with the seam strand of BamA. A ternary complex like this was first detected in this study. Based on these novel findings and the previous results, we proposed in this work the model of the BepA function in the LptD biogenesis. In this model, we suggested that the sequential movements of the a6 and a9 segments, which regulate the substrate binding and its proteolysis, respectively, enable the triage of LptD. We thus consider that our current results and the proposed model provide important clues to answer the above questions and substantial new insights into the mechanisms of the BepA functions and the LptD/OMP biogenesis. We believe that these findings and models are novel and worth publishing in a prestigious journal like eLife.

3b-iii) Figure 4 and 5: The models presented in Figures 4D, 5A, and 5B are essentially reproductions from Lee 2019, Daimon 2020, and Tomasek 2020. The main conceptual advance to the model is largely explained by Figure 4D and I'm not sure Figure 5 is warranted given its limited additional insight. I would perhaps move the entirety of Figure to the figure-supplements.

Figure 5A describes the possible sequential movement of the a6 and a9 segments, which will enable the triage of LptD. Figure 5B explains how the BepA-LptD-BAM ternary complex is formed during the LptD biogenesis and how the LptD intermediate is targeted to maturation or degradation. We think that these figures help readers understand our model and thus would like to keep them in the main part of the paper, if possible, although we are happy to move them to the figure-supplements if the editors and the reviewers recommend it.

Reviewer #1 (Recommendations for the authors):

1. This study is highly focused on BepA-LptD-BAM although the result can have a broad impact as a mode of protein quality control mechanisms in general. Readers may be lost in the midst of the detailed descriptions of experimental systems (M48 metalloproteases, BepA, LptD, LptD4213, etc.) and experimental results.

I recommend that authors emphasize broader implications of this work in the introduction and discussion. For example, readers would want to know more about:

Is LptD the only substrate of BepA?

Our previous (Figure 8B in Daimon et al. Mol. Microbiol. 106, 760-776, 2017; DOI:10.1111/mmi.13844) and the current (Figure 1—figure supplement 2A in this paper) results showed that BepA can degrade misassembled (and possibly mis- or un-folded) BamA in the DsurA strain. Thus, BepA might also act in the proteolytic quality control of other OMPs including BamA. It remains an open question whether BepA also acts in the assembly of some other OM proteins, like in the case of LptD. Systematic identification and analysis of the additional BepA substrates will be needed to know how generally BepA acts in the biogenesis/degradation of OM proteins.

These points were described in the revised text (lines 71-72 in p3, and lines 411–414 in p16).

What other biological processes is BepA involved?

Please see our response to the above question.

Is the involvement of the ternary complex (i.e., BAM, a OMP client, and a quality control enzyme) in LptD biogenesis a novel finding in the field?

A ternary complex formation among an assembly intermediate of an OM protein (EspP), a periplasmic chaperone (SurA/Skp), and the BAM complex has been suggested from biochemical studies including crosslinking, although the ternary complex was not directly detected (Ieva et al. PNAS, E383-391, 2011; doi:10.1073/pnas.1103827108). We here experimentally demonstrated the ternary complex formation for LptD, BepA, and the BAM complex. A similar client-chaperone-BAM ternary complex might be formed in the assembly of other OM proteins. This should be addressed in a future study.

The above points were described in the revised text (lines 292–297 in p12 and lines 409–412 in p16).

Would the proposed mechanism be generalizable to other BepA-mediated quality control of OMPs?

Please see our response to the above question.

2. Discussion is excellent. Nonetheless, it could be more easily conveyed to readers if it is divided into several subsections with proper headings.

Thank you for your valuable suggestion. We divided Discussion into subsections with proper headings according to the suggestion.

3. Clarification of the LptD mutant, LptD4213, is needed (in one sentence).

We added explanations of LptD4213 at two places ((1) lines 242–246 in p10, and (2) lines 353–357 in p14). Also, the properties of LptD4213 were described at other places ((3) lines 68–71 in p3, and (4) line 394 in p16). We suppose that these provide readers with sufficient information about LptD4213.

The details of the description are as follows:

1) It has been recently shown that LptD4213, a mutant form of LptD that has a short (23 amino acids) deletion in an extracellular loop (eL4) and is stalled on the BAM complex mimicking a late assembly complex (Lee et al., 2016), interacts with the seam region formed by the N- and C-terminal β-strands (β1 and β16, respectively) in the BamA barrel domain and forms a hetero-complex, in which the C-terminal β-signal of the LptD4213 was associated with the β1 strand of the BamA seam (Lee et al., 2019).

2) Notably, it has been reported that BepA expression can result in the degradation of a mutant form of LptD (LptD4213) that probably mimics a late-state assembly intermediate of LptD (Lee et al., 2019, 2016) that possesses a substantial degree of a higher order (b-barrel-like) structure and interacts with the BAM components (BamA and BamD), and LptE. The mechanism by which LptD4213 is degraded by BepA remains unclear, but its N-terminal region may interact with BepA.

3) While BepA promotes the LptDC to LptDNC conversion (chaperone-like function) (Narita et al., 2013), it also degrades the stalled or misassembled LptDC molecules that are generated due to an lptD mutation (lptD4213) or decreased availability of or weakened interaction with LptE (protease function) (Narita et al., 2013; Soltes et al., 2017).

4) Then, the folding (b-sheet formation) of the unstructured N-terminal region of the LptD β-barrel domain occurs to form a premature form of LptD with a substantially folded β-barrel domain and LptE within it, like LptD4213 (Lee et al., 2019, 2016)

Reviewer #2 (Recommendations for the authors):

1. Line 151-Line 160 and Figure 1D, the evidence of the pulse-chase experiments to examine the chaperone-like function of BepA is relatively weak. Since the N105P, A106P and F107P mutants disrupt the interaction with LptD and they have weaker proteolytic activities, these could result in LptDC accumulation to make the radio of LptDNC lower for these mutants. It's difficult to ignore the effects of the proteolytic activities in this assay.

Thank you for pointing out the problem of the data presentation. We re-analyzed the pule chase data. To examine only the chaperone-like activities (promotion of the LptDNC generation) without the possible effect of the LptD degradation by the residual proteolytic activities of the A106P and the F107P, the relative amounts of LptDNC at each chase point to the total LptD (LptDC + LptDNC) at the 5 min chase point were calculated and plotted as quantified data (new Figure 1D, right panel). The data clearly showed that the expression of WT BepA in the DbepA strain accelerated the maturation of LptD (that is, generation of LptDNC). Also, the A106P and the F107P mutant were mostly defective in the promotion of the LptD maturation. These results were described in the revised manuscript (lines 156–166 in p7). In addition, we previously showed that the expression of the protease-dead mutants, E137Q and H136R, significantly promoted the LptD maturation (please see Figure 4 in Narita et al. PNAS, 110, E3612–E3621, 2013; doi:10.1073/pnas.1312012110) and partially suppressed the OM-defective phenotypes of the bamB/bepA and bamE/bepA double-knockout strains (Figure 6 in Narita et al. PNAS, 110, E3612–E3621, 2013; doi:10.1073/pnas.1312012110) (note that in Figure 1D of the current work, the expression of the E137Q mutant only slightly promoted the LptD maturation. This would be ascribable to some difference in the experimental conditions including a lower expression level of the BepA mutant protein in the experiment for Figure 1D). These results collectively show that BepA possesses the chaperone-like activity (LptD maturation promoting activity) independent of its protease activity.

2. Does BepA only degrade LptDC not LptDNC?

Our previous results (Daimon et al. PNAS, 117, 27989-27996, 2020; DOI: https://doi.org/10.1073/pnas.2010301117) showed that BepA has little ability to degrade LptDC and LptDNC that are on the normal assembly pathway, but can degrade stalled or misassembled LptDC.

3. In Figure 4c and Figure 3B, a β-ME treatment control should be added to further confirm the crosslinking band of BepA×LptD×BamA.

For Figure 4C, as the crosslinker used (BMB) is not cleavable with β-ME, we did not include a β-ME treatment control. However, the presumed crosslinked product between BamA(S430C) and LptD(E773C) was generated in a BPB- and Cys-dependent manner (Figure 4—figure supplement 2A, see below), strongly suggesting that it was indeed the BMB-mediated crosslinked product between BamA and LptD. For Figure 3B, a β-ME treatment control has been shown in the original figure (the middle panel). Please note that in this experiment, the crosslinked products were purified by making use of the His-tag attached to LptD, and detected by anti-BepA immunoblotting. Thus, no crosslinked product was detected after the β-ME treatment. We noticed that the original Figure 4—figure supplement 2A contained several errors: aBepA should read aBamA and LptD-His10xBepA should read LptD-His10xBamA. We sincerely apologize to the editors and the reviewers for these mistakes.

4. Line 161-Line 165, it is not very clear why the authors performed Cys substitution for positions of N-105, A106, or F107? If they try to draw the conclusion that "the secondary structure of the edge-strand, but not the individual amino acid residues, is important for its function", they may mutate the individual position to different types of residues for experiments.

We chose a Cys substitution, because (i) the Cys mutants can be used in the disulfide crosslinking experiments, and (ii) a previous study strongly suggested that a Cys mutation does not affect the secondary structure of an edge-strand in another protease RseP (Akiyama et al. eLife, e08928, 2015; DOI:10.7554/eLife.08928). This was described in the revised text (lines 169–172 in p7).

5. For Figure 1, the authors should clearly label out the catalytic residues mentioned in the main text.

We annotated the catalytic residues in new Figure 1A to increase the clarity of the figure.

6. In general, it is a little bit hard to understand how come the edge-stand of BepA is able to directly contact with its substates as this this strand (β2) appears to be buried in the crystal structure of BepA. The authors should discuss this in the Discussion part.

We suppose that in both assembly and degradation pathways of LptD, an unknown signal(s), such as a specific interaction of BepA with the substrate and/or the BAM complex, induces a structural change in BepA (dislocation of the a6 loop covering the active site) to expose its active site and to enable the interaction between the LptD polypeptide and the edge-strand, whereas His-246 continues to repress the degradation of LptD by BepA. This was described in Discussion (lines 337–341 in p14). Please also see the model of Figure 5A.

7. The crosslinking assay revealed that BepA can interact with an LptD intermediate assembling on BAM complex. Does the dual function of BepA depend on the BAM complex?

We now have no direct evidence but think it possible that the BAM complex is involved in the switching of the dual functions of BepA through its direct interaction with BepA. This point was discussed in Discussion (lines 337–341 and 344 to 348 in p14, and lines 408–409). We would like to investigate this possibility in a future study.

Reviewer #3 (Recommendations for the authors):

My main criticism is the conclusion that the edge strand of BepA plays an active role to direct it (LptD) to on- and off- pathways. This is possible, but it would be important to show a mutation in the edge strand that could uncouple the chaperone and proteolytic activities of BepA. In both the A106P and F107P mutants, the loss in proteolytic activity is always accompanied by a loss in chaperone activity. I recognize that identifying such a mutant is not trivial, but the title of the paper implies that the edge strand plays a larger role in the functional switch in BepA.

As is, the manuscript describes in detail how BepA likely establishes the interaction with immature LptD on the Bam complex. but many of the general conclusions into the mechanism of LptD assembly have already been described elsewhere both by this group and others. As a result, the data in the work provides limited additional insight to the general mechanism of action of BepA.

The results by us and others have previously showed that BepA has both proteolytic and chaperone-like (that is, LptD-assembly promoting) activities. Although it has been easily expected that the edge-strand-mediated interaction with a substrate is important for its degradation, it is not known how BepA executes its chaperone-like function and how BepA distinguish the on- and off-pathway substrates. As described above, we showed that the normal interaction of the edge-strand of BepA with LptD is required not only for the degradation but also, unexpectedly, for the assembly promotion of this OMP substrate. Also, we demonstrated that BepA forms a ternary complex with LptD and the BAM complex in which BepA interacts with an unstructured N-terminal half of the LptD barrel domain and that the C-terminal region of LptD is associated with the seam strand of BamA. The ternary complex like this was first detected in this study. Based on these novel findings and the previous results, we proposed in this work the model of the BepA function in the LptD biogenesis. In this model, we suggested that the sequential movements of the a6 and a9 segments, which regulates the substrate binding and its proteolysis, respectively, enables the triage of LptD. We thus consider that our current results and the proposed models provide important clues to answer the above questions and substantial new insights into the mechanisms of the BepA functions and the LptD/OMP biogenesis. We believe that these findings and model are novel and worth publishing in a prestigious journal like eLife.

Detailed changes:

1) I would consider including a reference and an explanation describing the antibiotic sensitivity and complementation assays. Specifically, I think it would be helpful to explain how you can use erythromycin sensitivity to test BepA function in the antibiotic sensitivity assays and to explain how glucose/arabinose/IPTG regulate protein levels in the strains used for the complementation assays. These details may not be obvious to everyone and would be helpful for a more general readership. The description can be short and described in the figure captions.

According to the suggestions by the reviewer, we added brief explanations to the legends to Figure 1—figure supplement 3, Figure 2—figure supplement 2, and Figure 3—figure supplement 2 for how we can use erythromycin sensitivity to test the BepA function in the antibiotic sensitivity assays and for how glucose/arabinose/IPTG regulate protein levels in the strains used for the complementation assays.

2) All the structure figures look a little fuzzy and should be rendered at a higher resolution. This may be an artifact of the compression during submission, though.

We will upload high-resolution figures with the revised text.

3) Figure 1A (right): The 'catalytic residues' label is misspelled. The green in Regulatory His246 is also hard to read. I recommend changing the color.

We are sorry for the misspelling. We corrected the error. We also modified the color of His-246 to increase the clarity of Figurer 1A.

4) Figure 1D: I am weary about over interpreting the quantification of relative LptDNC levels, especially with the BepA F107P variant. I'm not sure if this is the best way to characterize BepA chaperone activity and the overall conclusions of this experiment still hold even if the quantification is removed.

Thank you for pointing out the problem of the data presentation. We re-analyzed the pule chase data. To examine only the chaperone-like activities (promotion of the LptDNC generation) without the possible effect of the LptD degradation by the residual proteolytic activities of the A106P and the F1107P, the relative amounts of LptDNC at each chase point to the total LptD (LptDC + LptDNC) at the 5 min chase point were calculated and plotted as quantified data (new Figure 1D, right panel). The data clearly showed that the expression of WT BepA in the DbepA strain accolated the maturation of LptD (that is, generation of LptDNC). Also, the A106P and the F107P mutant were mostly defective in the promotion of the LptD maturation. These results were described in the revised manuscript (lines 156–166 in p7). In addition, we previously showed that the expression of the protease-dead mutants, E137Q and H136R, significantly promoted the LptD maturation (please see Figure 4 in Narita et al. PNAS, 110, E3612–E3621, 2013; doi:10.1073/pnas.1312012110) and partially suppressed the OM-defective phenotypes of bamB/bepA and bamE/bepA double-knockout strains (Figure 6 in Narita et al. PNAS, 110, E3612–E3621, 2013; doi:10.1073/pnas.1312012110) (note that in Figure 1D of this paper, the expression of the E137Q mutant only slightly promoted that LptD maturation, possibly because of difference in the experimental condition including a lower expression level of the BepA mutant protein in this experiment). These results collectively show that BepA possesses the chaperone-like activity (LptD maturation promoting activity) independent of its protease activity.

5) Figure 2B and 3A: LptD pBPA E391 and Q393 both change the migration pattern of LptD rather noticeably. Do you know why this is the case? BPA substitution causes some changes at other positions, but these two in particular stand out. It is almost as if there is a deletion.

We confirmed that the genes for LptD pBPA E391 and Q393 have no deletion in their coding regions by sequencing the plasmids. We now do not know the exact reason why these proteins exhibited altered mobility, but we often encounter anomalous SDS gel mobility of pBPA-incorporated proteins and other missense mutant proteins. The anomalous mobility of the mutant proteins could be ascribed to changes in charges, and/or local structures that are formed even in the presence of SDS.

6) Figure 4: Figure 4C seems to suggest that the BepA antibody may also weakly cross react with BamA. If that's the case, then it is likely that the higher of the two molecular weight adducts labeled as BepAxLptD in both Figure 4A and 4B (lane 7) are actually LptDxBamA adducts. This band should be labeled with an asterisk instead.

As pointed out by the reviewer, our data (Figure 4C) indicate that the BepA antibody may also weakly cross-react with BamA. However, we think that the higher of the two molecular weight adducts labeled as BepAxLptD in Figure 4A and 4B are unlikely to be the LptDxBamA adducts for the following reason; while the anti-BamA immunoblotting in Figure 4A showed that the band intensities of the LptDxBamA adducts generated with LptD(D749pBPA) and LptD(Y331pBPA/D749pBPA) were much stronger than that generated with LptD(Y331pBPA), a band whose intensity was stronger than the higher of the two adducts generated with LptD(Y331pBPA) was not detected for LptD(D749pBPA) and LptD(Y331pBPA/D749pBPA) in ani-BepA immunoblotting. Instead, a band marked with an asterisk in the anti-BepA blots (in new Figure 4) possibly indicates an LptD-BamA crosslinked product that was detected due to the apparent cross-reactivity of the anti-BepA antibody with the LptDxBamA crosslinked product. We modified Figure 4 and explained this point in the legend to Figure 4.

7) Figure 4 and 5: The models presented in Figures 4D, 5A, and 5B are essentially reproductions from Lee 2019, Daimon 2020, and Tomasek 2020. The main conceptual advance to the model is largely explained by Figure 4D and I'm not sure Figure 5 is warranted given its limited additional insight. I would perhaps move the entirety of Figure to the figure-supplements.

Figure 5A describes the possible sequential movement of the a6 and a9 segments, which will enable the triage of LptD. Figure 5B explains how the BepA-LptD-BAM ternary complex is formed during the LptD biogenesis and how the LptD intermediate is targeted to maturation or degradation. We think that these figures help readers understand our model and thus would like to keep them in the main part of the paper, if possible, although we are happy to move them to the figure-supplements if the editors and the reviewers recommend it.

https://doi.org/10.7554/eLife.70541.sa2

Article and author information

Author details

  1. Ryoji Miyazaki

    Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
    Present address
    Division of Biological Science, Nara Institute of Science and Technology, Ikoma, Japan
    Contribution
    Conceptualization, Data curation, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Tetsuro Watanabe

    Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  3. Kohei Yoshitani

    Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Yoshinori Akiyama

    Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
    Contribution
    Conceptualization, Data curation, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    yakiyama@infront.kyoto-u.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4483-5408

Funding

Japan Society for the Promotion of Science (18H06047)

  • Ryoji Miyazaki

Japan Society for the Promotion of Science (19K21179)

  • Ryoji Miyazaki

Japan Society for the Promotion of Science (20K15715)

  • Ryoji Miyazaki

Japan Society for the Promotion of Science (15H01532)

  • Yoshinori Akiyama

Japan Society for the Promotion of Science (18H023404)

  • Yoshinori Akiyama

Institute for Fermentation, Osaka (Y-2020-02-027)

  • Ryoji Miyazaki

Nagase Science Technology Foundation

  • Yoshinori Akiyama

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank our laboratory members and S Narita for discussion and helpful advices. This work was supported by the Japan Society for the Promotion of Science KAKENHI Grants 18H06047, 19K21179, and 20K15715 (to RM), and 15H01532 and 18H023404 (to YA), and research grants from the Institute for Fermentation Y-2020-02-027 (to RM) and from the Nagase Science and Technology Foundation (to YA).

Senior Editor

  1. Gisela Storz, National Institute of Child Health and Human Development, United States

Reviewing Editor

  1. Heedeok Hong, Michigan State University, United States

Reviewers

  1. Heedeok Hong, Michigan State University, United States
  2. Yihua Huang

Publication history

  1. Preprint posted: March 28, 2021 (view preprint)
  2. Received: May 20, 2021
  3. Accepted: August 26, 2021
  4. Accepted Manuscript published: August 31, 2021 (version 1)
  5. Version of Record published: September 7, 2021 (version 2)

Copyright

© 2021, Miyazaki et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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