RESUMO
The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the ß-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.
Assuntos
Proteínas de Transporte , Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Peptidilprolil Isomerase , Trifosfato de Adenosina/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Peptidilprolil Isomerase/genética , Ligação Proteica , Domínios Proteicos , Dobramento de ProteínaRESUMO
Amyloid formation by α-synuclein (αSyn) occurs in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate αSyn amyloid fibril formation will not only provide mechanistic insight but may also reveal targets to prevent and treat disease. Previous investigations have identified several regions of αSyn to be important in the regulation of amyloid formation, including the non-amyloid-ß component (NAC), P1 region (residues 36 to 42), and residues in the C-terminal domain. Recent studies have also indicated the importance of the N-terminal region of αSyn for both its physiological and pathological roles. Here, the role of residues 2 to 7 in the N-terminal region of αSyn is investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (αSynΔN7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (αSynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length αSyn. Strikingly, fibril formation of αSynΔN7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to αSynWT. A Caenorhabditis elegans model also showed that αSynΔN7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of αSynWT::YFP. Together, the results demonstrate the involvement of residues 2 to 7 of αSyn in amyloid formation, revealing a target for the design of amyloid inhibitors that may leave the functional role of the protein in membrane binding unperturbed.
Assuntos
Amiloide , Caenorhabditis elegans , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/química , Amiloide/metabolismo , Caenorhabditis elegans/metabolismo , Animais , Humanos , Lipídeos/química , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologiaRESUMO
Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, each leaflet comprising a distinct lipid composition. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. In this study, we used cyclodextrin-mediated lipid exchange to generate liposomes with asymmetric bilayers and characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs), OmpA and BamA. We found that excess negative charge in the outer leaflet of a liposome impedes their insertion and folding, while excess negative charge in the inner leaflet accelerates their folding relative to symmetric liposomes with the same membrane composition. Using molecular dynamics, mutational analysis and bioinformatics, we identified a positively charged patch critical for folding and stability. These results rationalize the well-known 'positive-outside' rule of OMPs and suggest insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.
Assuntos
Proteínas de Escherichia coli , Lipossomos , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas de Escherichia coli/química , Proteínas da Membrana Bacteriana Externa/química , Lipídeos , Bicamadas Lipídicas/químicaRESUMO
Correct folding of outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria depends on delivery of unfolded OMPs to the ß-barrel assembly machinery (BAM). How unfolded substrates are presented to BAM remains elusive, but the major OMP chaperone SurA is proposed to play a key role. Here, we have used hydrogen deuterium exchange mass spectrometry (HDX-MS), crosslinking, in vitro folding and binding assays and computational modelling to show that the core domain of SurA and one of its two PPIase domains are key to the SurA-BAM interaction and are required for maximal catalysis of OMP folding. We reveal that binding causes changes in BAM and SurA conformation and/or dynamics distal to the sites of binding, including at the BamA ß1-ß16 seam. We propose a model for OMP biogenesis in which SurA plays a crucial role in OMP delivery and primes BAM to accept substrates for folding.
Assuntos
Proteínas de Escherichia coli , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Periplasma/metabolismo , Dobramento de ProteínaRESUMO
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/ultraestruturaRESUMO
BACKGROUND: Malaria kills over 400,000 people each year and nearly half the world's population live in at-risk areas. Progress against malaria has recently stalled, highlighting the need for developing novel therapeutics. The parasite haemoglobin degradation pathway, active in the blood stage of the disease where malaria symptoms and lethality manifest, is a well-established drug target. A key enzyme in this pathway is the papain-type protease falcipain-2. METHODS: The crystallographic structure of falcipain-2 at 3.45 Å resolution was resolved in complex with an (E)-chalcone small-molecule inhibitor. The falcipain-2-(E)-chalcone complex was analysed with reference to previous falcipain complexes and their similarity to human cathepsin proteases. RESULTS: The (E)-chalcone inhibitor binds falcipain-2 to the rear of the substrate-binding cleft. This is the first structure of a falcipain protease where the rear of the substrate cleft is bound by a small molecule. In this manner, the (E)-chalcone inhibitor mimics interactions observed in protein-based falcipain inhibitors, which can achieve high interaction specificity. CONCLUSIONS: This work informs the search for novel anti-malaria therapeutics that target falcipain-2 by showing the binding site and interactions of the medically privileged (E)-chalcone molecule. Furthermore, this study highlights the possibility of chemically combining the (E)-chalcone molecule with an existing active-site inhibitor of falcipain, which may yield a potent and selective compound for blocking haemoglobin degradation by the malaria parasite.