Inhibition of the β-barrel assembly machine by a peptide that binds BamD

Hagan, Christine L.; Wzorek, Joseph S.; Kahne, Daniel

doi:10.1073/pnas.1415955112

Cited by 112 publications

(157 citation statements)

References 47 publications

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“…Furthermore, we can speculate that P5 is involved in recognition of the C-terminal motif of substrate OMPs (Robert et al, 2006), transiently accommodating the negatively charged C terminus within its dynamic electrostatic network. Interestingly, also BamD has been implied in binding of the C-terminal motif (Albrecht and Zeth, 2011;Hagan et al, 2015;Sandoval et al, 2011) and further studies will have to elucidate the precise interplay between P5, BamD, and the substrate (Figures 5C-3).…”

Section: Discussionmentioning

confidence: 99%

Conformational Plasticity of the POTRA 5 Domain in the Outer Membrane Protein Assembly Factor BamA

et al. 2015

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BamA is the main component of the β-barrel assembly machinery (BAM) that folds and inserts outer membrane proteins in Gram-negative bacteria. Crystal structures have suggested that this process involves conformational changes in the transmembrane β-barrel of BamA that allow for lateral opening, as well as large overall rearrangements of its periplasmic POTRA domains. Here, we identify local dynamics of the BamA POTRA 5 domain by solution and solid-state nuclear magnetic resonance. The protein region undergoing conformational exchange is highly conserved and contains residues critical for interaction with BamD and correct β-barrel assembly in vivo. We show that mutations known to affect the latter processes influence the conformational equilibrium, suggesting that the plasticity of POTRA 5 is related to its interaction with BamD and possibly to substrate binding. Taken together, a view emerges in which local protein plasticity may be critically involved in the different stages of outer membrane protein folding and insertion.

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Section: Discussionmentioning

confidence: 99%

Conformational Plasticity of the POTRA 5 Domain in the Outer Membrane Protein Assembly Factor BamA

et al. 2015

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Section: Overall Architecture Of the Bam Complexmentioning

confidence: 99%

“…One of the functions of BamD is a substrate recognition (34). BamD binds unfolded OMP substrates, and the binding site lays within the C-terminal OMP sequences which closely resembles the mitochondrial β signal (33; 34). The β signal targets mitochondrial OMPs to a homologous Sam complex, and the BamD analog, Sam35, is involved in this recognition (55).…”

Section: Overall Architecture Of the Bam Complexmentioning

confidence: 99%

“…The β signal targets mitochondrial OMPs to a homologous Sam complex, and the BamD analog, Sam35, is involved in this recognition (55). Ability to recognize the substrates is an essential function of BamD, and the peptides containing the β signal, which bind BamD, are toxic because they inhibit β barrel assembly (34). However, the function of BamD is not limited to the initial substrate recognition, because BamD remains closely associated with the substrate throughout entire OMP assembly and must play an active role during this process (40; 56).…”

Section: Overall Architecture Of the Bam Complexmentioning

confidence: 99%

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Outer Membrane Biogenesis

Konovalova

Kahne²,

Silhavy

2017

Annu. Rev. Microbiol.

253

229

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The hallmark of Gram-negative bacteria and organelles such as mitochondria and chloroplasts is the presence of an outer membrane (OM). In bacteria such as Escherichia coli, the OM is a unique asymmetric lipid bilayer with lipopolysaccharide (LPS) in the outer leaflet. Integral, transmembrane proteins assume a β-barrel structure (OMPs) and their assembly is catalyzed by the heteropentomeric Bam complex containing the OMP BamA and four lipoproteins, BamB-E. How the Bam complex assembles a great diversity of OMPs into a membrane without an obvious energy source is a particularly challenging problem because folding intermediates are predicted to be unstable in either an aqueous or a hydrophobic environment. Two models have been put forward, the budding model based largely on structural data, and the BamA-assisted model based on genetic and biochemical studies. Here we offer a critical discussion of the pros and cons of each.

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“…Because Bam also contains an essential lipoprotein, BamD (5, 7), we tested if this protein, like BamA (30)(31)(32)(33), is involved in the assembly of barrel substrates (34,35). We performed the same cross-linking experiments as for BamA except in cells expressing His-tagged BamD (Fig.…”

Section: Significancementioning

confidence: 99%

Characterization of a stalled complex on the β-barrel assembly machine

Lee

Xue

Wzorek

et al. 2016

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

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112

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The assembly of β-barrel proteins into membranes is mediated by an evolutionarily conserved machine. This process is poorly understood because no stable partially folded barrel substrates have been characterized. Here, we slowed the folding of the Escherichia coli β-barrel protein, LptD, with its lipoprotein plug, LptE. We identified a late-stage intermediate in which LptD is folded around LptE, and both components interact with the two essential β-barrel assembly machine (Bam) components, BamA and BamD. We propose a model in which BamA and BamD act in concert to catalyze folding, with the final step in the process involving closure of the ends of the barrel with release from the Bam components. Because BamD and LptE are both soluble proteins, the simplest model consistent with these findings is that barrel folding by the Bam complex begins in the periplasm at the membrane interface.outer membrane | Bam complex | β-barrel | protein folding T he assembly of β-barrel membrane proteins into the outer membrane (OM) of Gram-negative bacteria, mitochondria, and chloroplasts is facilitated by conserved cellular machinery (1-4). The β-barrel assembly machine (Bam) folds and inserts integral membrane proteins into the OM of Gram-negative organisms (5). Bam is a five-protein complex consisting of the essential protein BamA, a β-barrel itself, and four lipoproteins, BamB, -C, -D, and -E, of which only BamD is essential (4-8). The Bam complex recognizes a large number of different substrates, but how each component catalyzes the folding and insertion of such structurally diverse substrates is unclear.How β-barrels are assembled into membranes is not obvious. Where and how folding occurs is unclear because intermediates could contain both exposed polar amides and hydrophobic residues until the barrel has completed its fully hydrophobic exterior. By contrast, α-helical membrane proteins have internally satisfied hydrogen bonds, making stepwise assembly from stable secondary structural elements possible. Although Bam has been shown to accelerate membrane β-barrel assembly (9-11), the transient nature of folding intermediates has made accumulating such discrete species for characterization difficult (12-15). If structurally defined folding intermediates were to exist long enough for characterization, they could reveal crucial aspects of the folding process.Here, we studied the assembly of an essential, slow-folding β-barrel, LptD. LptD is one of two components of the OM translocon that transports lipopolysaccharide to the cell surface (16-18). The other component, LptE, is a lipoprotein that forms a plug inside the LptD barrel (19)(20)(21)(22). LptD also contains two disulfide bonds (23), and its assembly involves the formation of consecutive disulfide bonds that after barrel folding rearrange to form nonconsecutive disulfide bonds (24). The assembly of LptD is orders-ofmagnitude slower (∼20 min versus seconds) than that of other barrel substrates (24-26). Because of the slow rate of folding and our ability to use oxidation sta...

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Inhibition of the β-barrel assembly machine by a peptide that binds BamD

Cited by 112 publications

References 47 publications

Conformational Plasticity of the POTRA 5 Domain in the Outer Membrane Protein Assembly Factor BamA

Conformational Plasticity of the POTRA 5 Domain in the Outer Membrane Protein Assembly Factor BamA

Outer Membrane Biogenesis

Characterization of a stalled complex on the β-barrel assembly machine

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