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

RESUMO

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.


Assuntos
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
2.
Commun Biol ; 3(1): 766, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318620

RESUMO

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.


Assuntos
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
3.
Protein Sci ; 29(8): 1851-1857, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32557917

RESUMO

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yield inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly populated "excited" states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein, and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualization of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication.


Assuntos
Modelos Moleculares , Proteínas/química , Software , Conformação Proteica
4.
Nat Commun ; 11(1): 2155, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358557

RESUMO

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Proteínas de Transporte/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Espectrometria de Massas , Chaperonas Moleculares/genética , Peptidilprolil Isomerase/genética , Ligação Proteica
5.
Nat Commun ; 11(1): 1816, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286330

RESUMO

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.


Assuntos
Agregados Proteicos , Sequência de Aminoácidos , Substituição de Aminoácidos , Regiões Determinantes de Complementaridade/química , Escherichia coli/metabolismo , Humanos , Imunoglobulina G/química , Viabilidade Microbiana , Mutação/genética , Anticorpos de Cadeia Única/química , beta-Lactamases/química
6.
J Mol Biol ; 431(6): 1267-1283, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30716334

RESUMO

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.


Assuntos
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
7.
BMC Biol ; 15(1): 123, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29268734

RESUMO

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the ß-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bactérias Gram-Negativas/metabolismo , Metabolismo Energético , Dobramento de Proteína
8.
J Mol Biol ; 429(23): 3776-3792, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-28919234

RESUMO

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.


Assuntos
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
9.
Nat Commun ; 7: 12865, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27686148

RESUMO

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.

10.
Nat Struct Mol Biol ; 23(9): 786-793, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27455461

RESUMO

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry-mass spectrometry (IMS-MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Cinética , Chaperonas Moleculares/fisiologia , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína
11.
ACS Nano ; 9(9): 8811-21, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26284289

RESUMO

Single-molecule force spectroscopy by atomic force microscopy exploits the use of multimeric protein constructs, namely, polyproteins, to decrease the impact of nonspecific interactions, to improve data accumulation, and to allow the accommodation of benchmarking reference domains within the construct. However, methods to generate such constructs are either time- and labor-intensive or lack control over the length or the domain sequence of the obtained construct. Here, we describe an approach that addresses both of these shortcomings that uses Gibson assembly (GA) to generate a defined recombinant polyprotein rapidly using linker sequences. To demonstrate the feasibility of this approach, we used GA to make a polyprotein composed of alternating domains of I27 and TmCsp, (I27-TmCsp)3-I27)(GA), and showed the mechanical fingerprint, mechanical strength, and pulling speed dependence are the same as an analogous polyprotein constructed using the classical approach. After this benchmarking, we exploited this approach to facilitiate the mechanical characterization of POTRA domain 2 of BamA from E. coli (EcPOTRA2) by assembling the polyprotein (I27-EcPOTRA2)3-I27(GA). We show that, as predicted from the α + ß topology, EcPOTRA2 domains are mechanically robust over a wide range of pulling speeds. Furthermore, we identify a clear correlation between mechanical robustness and brittleness for a range of other α + ß proteins that contain the structural feature of proximal terminal ß-strands in parallel geometry. We thus demonstrate that the GA approach is a powerful tool, as it circumvents the usual time- and labor-intensive polyprotein production process and allows for rapid production of new constructs for single-molecule studies. As shown for EcPOTRA2, this approach allows the exploration of the mechanical properties of a greater number of proteins and their variants. This improves our understanding of the relationship between structure and mechanical strength, increasing our ability to design proteins with tailored mechanical properties.


Assuntos
Peptídeos/química , Poliproteínas/química , Multimerização Proteica , Escherichia coli/química , Microscopia de Força Atômica , Poliproteínas/ultraestrutura , Estrutura Terciária de Proteína
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