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Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105


The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
Commun Biol ; 3(1): 766, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318620


The ß-barrel assembly machinery (BAM) catalyses the folding and insertion of ß-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The ß-barrel domain of BamA is in a 'lateral open' conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM's lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid 'disruptase' activity of BAM, suggested to be an important part of its functional mechanism.

Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas , Lipídeos , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Nanoestruturas , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/metabolismo , Catálise , Complexos Multiproteicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteolipídeos/metabolismo
Biochim Biophys Acta Biomembr ; 1862(5): 183192, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31945320


Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications - particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function.

Maleatos/química , Lipídeos de Membrana/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Poliestirenos/química , Microscopia Crioeletrônica/métodos , Detergentes , Escherichia coli/química , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/química , Gotículas Lipídicas/química , Espectrometria de Massas/métodos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Polímeros/química
J Mol Biol ; 431(6): 1267-1283, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30716334


SurA is a conserved ATP-independent periplasmic chaperone involved in the biogenesis of outer-membrane proteins (OMPs). Escherichia coli SurA has a core domain and two peptidylprolyl isomerase (PPIase) domains, the role(s) of which remain unresolved. Here we show that while SurA homologues in early proteobacteria typically contain one or no PPIase domains, the presence of two PPIase domains is common in SurA in later proteobacteria, implying an evolutionary advantage for this domain architecture. Bioinformatics analysis of >350,000 OMP sequences showed that their length, hydrophobicity and aggregation propensity are similar across the proteobacterial classes, ruling out a simple correlation between SurA domain architecture and these properties of OMP sequences. To investigate the role of the PPIase domains in SurA activity, we deleted one or both PPIase domains from E.coli SurA and investigated the ability of the resulting proteins to bind and prevent the aggregation of tOmpA (19 kDa) and OmpT (33 kDa). The results show that wild-type SurA inhibits the aggregation of both OMPs, as do the cytoplasmic OMP chaperones trigger factor and SecB. However, while the ability of SurA to bind and prevent tOmpA aggregation does not depend on its PPIase domains, deletion of even a single PPIase domain ablates the ability of SurA to prevent OmpT aggregation. The results demonstrate that the core domain of SurA endows its generic chaperone ability, while the presence of PPIase domains enhances its chaperone activity for specific OMPs, suggesting one reason for the conservation of multiple PPIase domains in SurA in proteobacteria.

Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Peptídeo Hidrolases/metabolismo , Peptidilprolil Isomerase/química , Domínios Proteicos , Proteínas da Membrana Bacteriana Externa/química , Fenômenos Biofísicos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Proteobactérias/metabolismo , Deleção de Sequência
J Mol Biol ; 429(23): 3776-3792, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-28919234


The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the ß-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA ß-barrel domain, but is greater when full-length BamA is present, indicating that both the ß-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro.

Proteínas da Membrana Bacteriana Externa/química , Membrana Celular/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Periplasma/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/metabolismo , Biocatálise , Membrana Celular/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Lipossomos/metabolismo , Simulação de Dinâmica Molecular
Nat Commun ; 7: 12865, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27686148


The ß-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA-E), which is essential for viability in E. coli. BAM promotes the folding and insertion of ß-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a 'lateral gating' motion of the ß-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.