Membrane Defects Accelerate Outer Membrane β-Barrel Protein Folding

Danoff, Emily J.; Fleming, Karen G.

doi:10.1021/bi501443p

Cited by 53 publications

(70 citation statements)

References 24 publications

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“…Remarkably, EspP∆5 folded into proteoliposomes prepared with the gel phase lipid DPPC only slightly less efficiently than proteoliposomes prepared with the corresponding fluid phase lipid POPC. This is a striking result given that the folding of OMPs into pure gel phase liposomes has not been observed (25,28,32). We also found that EspP∆5 folded slightly less efficiently into proteoliposomes prepared with a mixture of POPC and POPE, POPG or CL than those prepared with POPC alone (Fig.…”

Section: The Bam Complex Catalyzes Efficient Folding Of Espp Into a Wmentioning

confidence: 52%

“…Nevertheless, the results suggest that the ability of OMPs to fold is encoded within their amino acid sequence. The results also indicate that properties of lipid bilayers including fluidity (32,33), thickness (33), and head group charge or type (34) strongly affect the folding of many different β-barrel proteins. Although individual OMPs differ in their propensity to fold in different lipid environments (26,27), thin, fluidphase bilayers with charge-neutral lipid head groups (33) generally facilitate OMP folding, while naturally-occurring zwitterionic or negatively-charged lipid head groups inhibit or sometimes even preclude folding altogether (26).…”

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confidence: 86%

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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: The Bam Complex Catalyzes Efficient Folding Of Espp Into a Wmentioning

confidence: 52%

mentioning

confidence: 86%

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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“…Reductions in temperature will also further decrease the fluidity of the characteristically rigid OM (15,16), which could exacerbate assembly defects of certain classes of proteins. The kinetics of OMP folding into a membrane is strongly influenced by the physical properties of the membrane, and temperature can strongly affect the biophysical properties of the OM, leading to changes in the rate of OMP folding with temperature (17)(18)(19)(20). Alternatively, or in addition, it seems likely that there is an increased requirement for periplasmic proteolysis in the BamA/BamD down mutant strains.…”

Section: Discussionmentioning

confidence: 99%

Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

2016

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The biogenesis of the outer membrane (OM) of Escherichia coli is a conserved and vital process. The assembly of integral ␤-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric ␤-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, lowabundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations in bamD that lower levels of BamD 10-fold and >25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wildtype BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM. IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral ␤-barrel proteins (OMPs) are assembled by the ␤-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins. The assembly of integral ␤-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria is essential for cell growth and viability. OMP targeting and OM integration depend on an assembly pathway comprising semiredundant periplasmic chaperones and the heteropentameric OM-associated ␤-barrel assembly machine (Bam). The primary constituents of the periplasmic chaperone network in Escherichia coli are the proline isomerases SurA and FkpA, the bifunctional protease/chaperone DegP, and the prefoldin-like chaperone Skp. The Bam complex is composed of BamA (itself a ␤-barrel) and four associated lipoproteins, BamB, BamC, BamD, and BamE (1-3).Although OMPs require the Bam complex for folding into the OM, the individ...

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“…This process continues until the barrel is fully formed and membrane integrated. Such spontaneous folding can only occur in thin membranes made up by the short acyl chain lipids and at a temperature at or above phase transition, demonstrating the requirement of a membrane defect for an insertion (15). Native E. coli phospholipids, such as phosphoethanolamine (PE) and or phosphoglycerol (PG), do not support spontaneous OMP folding and hence a model was proposed that the Bam complex functions to reduce the kinetic barrier of membrane insertion by either creating a membrane defect and/or stabilizing certain transition conformations of the OMP at the membrane (28).…”

Section: Models For the β-Barrel Assemblymentioning

confidence: 99%

Outer Membrane Biogenesis

Konovalova

Kahne²,

Silhavy

2017

Annu. Rev. Microbiol.

247

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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Membrane Defects Accelerate Outer Membrane β-Barrel Protein Folding

Cited by 53 publications

References 24 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

Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

Outer Membrane Biogenesis

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