Structure and function of BamE within the outer membrane and the β‐barrel assembly machine

Knowles, Timothy J.; Browning, Douglas F.; Jeeves, Mark; Maderbocus, Riyaz; Rajesh, Sundaresan; Sridhar, Pooja; Manoli, Eleni; Emery, Danielle; Sommer, Ulf; Spencer, A. J.; Leyton, Denisse L.; Squire, Derrick J. P.; Chaudhuri, Roy R.; Viant, Mark R.; Cunningham, Adam F.; Henderson, Ian R.; Overduin, Michael

doi:10.1038/embor.2010.202

Cited by 88 publications

(95 citation statements)

References 31 publications

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“…Thus, while BamA alone can catalyze limited OMP assembly, our results imply that the Bam complex lipoproteins play an important role in promoting folding in a native lipid context. Although evidence that the BamA POTRA domains interact with negatively-charged lipids (66) and that BamE binds to PG (13,67) suggests that the recruitment of specific lipids stabilizes lipoprotein binding or facilitates OMP folding, our finding that the lipid environment does not significantly affect OMP assembly challenges the hypothesis that the Bam complex functions by exploiting the anionic membrane surface. Likewise, our results indicate that CL, which appears to play a stimulatory role in the assembly of mitochondrial β-barrels (68), does not facilitate Bam complex-mediated folding.…”

Section: Discussioncontrasting

confidence: 54%

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Hussain

Bernstein

2018

Journal of Biological Chemistry

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Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "b-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively, but often requires nonphysiological conditions and time scales and is strongly influenced by properties of the lipid bilayer including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the b-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA and another model protein (OmpG) folded at significantly different rates and that an a-helix embedded inside the EspP b-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding. __________________________ Gram-negative bacteria, a class that includes many pathogenic and emerging antibiotic-resistant organisms, are bound by a double cell membrane. Most of the proteins that reside in the outer membrane (OM) 2 , which serves as a robust barrier, have a distinctive "b-barrel" architecture. A bbarrel is essentially a b-sheet rolled into a closed cylinder that has a hydrophobic exterior and a hydrophilic interior and that is held together by hydrogen bonds between the first and last bstrands. The b-barrel scaffold is found exclusively in the OM of bacteria and organelles of bacterial origin. OM proteins (OMPs) mediate many different functions vital for bacterial survival and range considerably in size from 8-26 b-strands (1, 2). Some OMPs form oligomers (3), while others contain a segment that is embedded inside the bbarrel or a separately folded domain that is displayed on either the periplasmic or extracellular side of the OM (1).OMPs must first traverse the inner membrane (IM) and the periplasmic space, which is devoid of ATP (4), before they attain their final structure in the OM. After OMPs are transported across the IM in an unfolded conformation through the Sec translocon, a variety of periplasmic chaperones including SurA, Skp, and DegP prevent their aggregation in the periplasm (5-7). Integration into the OM is then catalyzed by the heterooligomeric β-barrel assembly machinery (Bam) complex (8,9). In E. coli, the Bam complex is composed of BamA, a b-barrel protein that contains five http://www.jbc.org/cgi

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Section: Discussioncontrasting

confidence: 54%

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Hussain

Bernstein

2018

Journal of Biological Chemistry

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“…of ϳ2.5 Å, with a reasonable agreement in the ␤-sheet part but significant deviation in the helix and loop structures (supplemental Fig. S7A) (39,45).…”

Section: Resultsmentioning

confidence: 88%

“…Notably, the interface between these two monomers accounts to a significant ϳ1200 Å 2 . The dimer architecture and irreversible monomer-dimer equilibrium are indicative of a three-dimensional domain swapping of the N terminus exchanging with the C-terminal domain (39,44,45) (Fig. 5, A and B and supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, this structure was also solved by NMR methods, but a monomer was found, although with a similar topology as the dimeric species (40,45). Because the equilibrium is irreversible and domain swapping in many proteins was shown to be activating a protein complex, it seems more likely that the dimer is the active form, whereas the monomer is inactive (44).…”

Section: Discussionmentioning

confidence: 99%

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Structural Basis of Outer Membrane Protein Biogenesis in Bacteria

Albrecht

Zeth

2011

Journal of Biological Chemistry

104

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In Escherichia coli, a multicomponent BAM (␤-barrel assembly machinery) complex is responsible for recognition and assembly of outer membrane ␤-barrel proteins. The functionality of BAM in protein biogenesis is mainly orchestrated through the presence of two essential components, BamA and BamD. Here, we present crystal structures of four lipoproteins (BamB-E). Monomeric BamB and BamD proteins display scaffold architectures typically implied in transient protein interactions. BamB is a ␤-propeller protein comprising eight WD40 repeats. BamD shows an elongated fold on the basis of five tetratricopeptide repeats, three of which form the scaffold for protein recognition. The rod-shaped BamC protein has evolved through the gene duplication of two conserved domains known to mediate protein interactions in structurally related complexes. By contrast, the dimeric BamE is formed through a domain swap and indicates fold similarity to the ␤-lactamase inhibitor protein family, possibly integrating cell wall stability in BAM function. Structural and biochemical data show evidence for the specific recognition of amphipathic sequences through the tetratricopeptide repeat architecture of BamD. Collectively, our data advance the understanding of the BAM complex and highlight the functional importance of BamD in amphipathic outer membrane ␤-barrel protein motif recognition and protein delivery.Gram-negative bacteria are surrounded by two membranes, an inner membrane and the protective outer membrane (OM) 2 layer. The outer membrane architecture is highly asymmetric and composed by integral outer membrane ␤-barrel proteins, lipoproteins, lipopolysaccharides, and phospholipids (1). These components are entirely synthesized in the cytoplasm, translocated over the inner membrane, and finally delivered through the periplasm to the outer membrane by specific shuttle factors that transport their cargo to membrane receptor complexes (1, 2). In Escherichia coli, lipoproteins are targeted to the OM through the LolABCDE complex system via a series of membrane and periplasmic transfer steps (3, 4). Integral OMPs are translocated by the secretion (Sec) machinery and stabilized against premature precipitation or mislocalization in the periplasm by the three major chaperones PpiD, Skp, and SurA (4 -7). These chaperones presumably act sequentially with PpiD at an early stage, early after the OMP release into the periplasm (5). This initial rescue event is followed by Skp and SurA chaperoning at a later stage (8). Various experimental studies describing OMP folding through SurA show the particular importance of this chaperone. Phage display combined with peptide binding studies indicated SurA interactions with amphipathic OMP peptides (9, 10). Furthermore, the importance of SurA in vivo is underlined by the observation that E. coli cells being deleted in surA exhibit reduced levels of folded OmpA, OmpC, OmpF, and LamB porins (11).After the unfolded outer membrane protein is delivered through the periplasmic space, the final steps are the tetherin...

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“…However, the mechanism by which β-barrel assembly proceeds is only beginning to be elucidated. Structures of the components of the bacterial β-barrel assembly machine (BamA-E) have been determined recently, and several hypotheses about how they facilitate the assembly of its substrates have been proposed (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The β-barrel component BamA contains a kinked β-strand, which might allow substrate proteins to be inserted by passing laterally from the lumen of the BamA β-barrel into the hydrophobic membrane (18).…”

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confidence: 99%

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

Hagan

Wzorek

Kahne

2015

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

112

145

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The protein complex that assembles integral membrane β-barrel proteins in the outer membranes of Gram-negative bacteria is an attractive target in the development of new antibiotics. This complex, the β-barrel assembly machine (Bam), contains two essential proteins, BamA and BamD. We have identified a peptide that inhibits the assembly of β-barrel proteins in vitro by characterizing the interaction of BamD with an unfolded substrate protein. This peptide is a fragment of the substrate protein and contains a conserved amino acid sequence. We have demonstrated that mutations of this sequence in the full-length substrate protein impair the protein's assembly, implying that BamD's interaction with this sequence is an important part of the assembly mechanism. Finally, we have found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant. Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria.β-barrel | outer membrane | protein folding | Bam complex | inhibition

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Structure and function of BamE within the outer membrane and the β‐barrel assembly machine

Cited by 88 publications

References 31 publications

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Structural Basis of Outer Membrane Protein Biogenesis in Bacteria

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

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