RESUMEN
The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Ribosomas , Microscopía por Crioelectrón/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Unión al GTP/metabolismo , Microfluídica/métodos , Ribosomas/metabolismo , Dióxido de Silicio/análisisRESUMEN
Transcription-coupled repair (TCR), discovered as preferential nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers located in transcribed mammalian genes compared to those in nontranscribed regions of the genome, is defined as faster repair of the transcribed strand versus the nontranscribed strand in transcribed genes. The phenomenon, universal in model organisms including Escherichia coli, yeast, Arabidopsis, mice, and humans, involves a translocase that interacts with both RNA polymerase stalled at damage in the transcribed strand and nucleotide excision repair proteins to accelerate repair. Drosophila, a notable exception, exhibits TCR but lacks an obvious TCR translocase. Mutations inactivating TCR genes cause increased damage-induced mutagenesis in E. coli and severe neurological and UV sensitivity syndromes in humans. To date, only E. coli TCR has been reconstituted in vitro with purified proteins. Detailed investigations of TCR using genome-wide next-generation sequencing methods, cryo-electron microscopy, single-molecule analysis, and other approaches have revealed fascinating mechanisms.
Asunto(s)
Escherichia coli , Transcripción Genética , Humanos , Animales , Ratones , Escherichia coli/genética , Escherichia coli/metabolismo , Microscopía por Crioelectrón , Reparación del ADN , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Mamíferos/genéticaRESUMEN
In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.
Asunto(s)
Proteínas de Escherichia coli , Transcripción Genética , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Ribosomas/metabolismo , Proteínas de Escherichia coli/genéticaRESUMEN
Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while "riding" the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance "vehicle" for detecting the most frequently occurring replication errors.
Asunto(s)
Reparación del ADN , ARN Polimerasas Dirigidas por ADN , Escherichia coli , Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Ribonucleótidos/metabolismoRESUMEN
Transmembrane ß barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the ß barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model ß barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "ß signal" motif of EspP to correctly orient ß strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated ß sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that ß sheets progressively fold toward BamA to form a ß barrel. Along with in vivo experiments that tracked ß barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate ß barrel folding.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/ultraestructura , Pliegue de Proteína , Proteínas de la Membrana Bacteriana Externa/metabolismo , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
Type 1 secretion systems (T1SSs) are widespread in pathogenic Gram-negative bacteria, extruding protein substrates following synthesis of the entire polypeptide. The Escherichia coli hemolysin A secretion system has long been considered a prototype in structural and mechanistic studies of T1SSs. Three membrane proteins-an inner membrane ABC transporter HlyB, an adaptor protein HlyD, and an outer membrane porin TolC-are required for secretion. However, the stoichiometry and structure of the complex are unknown. Here, cryo-electron microscopy (cryo-EM) structures determined in two conformations reveal that the inner membrane complex is a hetero-dodecameric assembly comprising three HlyB homodimers and six HlyD subunits. Functional studies indicate that oligomerization of HlyB and HlyD is essential for protein secretion and that polypeptides translocate through a canonical ABC transporter pathway in HlyB. Our data suggest that T1SSs entail three ABC transporters, one that functions as a protein channel and two that allosterically power the translocation process.
Asunto(s)
Proteínas de Escherichia coli , Proteínas Hemolisinas , Transportadoras de Casetes de Unión a ATP/metabolismo , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.
Asunto(s)
ADN Mitocondrial , Enfermedades Mitocondriales , Animales , Sistemas CRISPR-Cas , Citosina/metabolismo , ADN Mitocondrial/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Edición Génica , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , PurinasRESUMEN
Bacterial cytoplasmic membrane vesicles provide a unique experimental system for studying active transport. Vesicles are prepared by lysis of osmotically sensitized cells (i.e., protoplasts or spheroplasts) and comprise osmotically intact, unit-membrane-bound sacs that are approximately 0.5-1.0 µm in diameter and devoid of internal structure. Their metabolic activities are restricted to those provided by the enzymes of the membrane itself, and each vesicle is functional. The energy source for accumulation of a particular substrate can be determined by studying which compounds or experimental conditions drive solute accumulation, and metabolic conversion of the transported substrate or the energy source is minimal. These properties of the vesicle system constitute a considerable advantage over intact cells, as the system provides clear definition of the reactions involved in the transport process. This discussion is not intended as a general review but is concerned with respiration-dependent active transport in membrane vesicles from Escherichia coli. Emphasis is placed on experimental observations demonstrating that respiratory energy is converted primarily into work in the form of a solute concentration gradient that is driven by a proton electrochemical gradient, as postulated by the chemiosmotic theory of Peter Mitchell.
Asunto(s)
Vesículas Citoplasmáticas/metabolismo , Escherichia coli/metabolismo , Biología Molecular/historia , Transporte Biológico , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Membrana Celular/efectos de los fármacos , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Ácido Láctico/metabolismo , Masculino , Estados UnidosRESUMEN
Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Imagenología Tridimensional , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Escherichia coli/enzimología , Escherichia coli/metabolismo , Espectrometría de Masas , Simulación de Dinámica Molecular , Presión Osmótica , Fosforilación , Proteolisis , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Estrés FisiológicoRESUMEN
PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Endocitosis , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestructura , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Liposomas Unilamelares/metabolismoRESUMEN
All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.
Asunto(s)
Cromosomas Bacterianos/química , Escherichia coli/metabolismo , Conformación de Ácido Nucleico , Solventes/química , Transcripción Genética , Aminoglicósidos/farmacología , Simulación por Computador , ADN Bacteriano/química , Difusión , Escherichia coli/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Tamaño de la Partícula , ARN Bacteriano/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructura , Transcripción Genética/efectos de los fármacosRESUMEN
Manipulation of individual molecules with optical tweezers provides a powerful means of interrogating the structure and folding of proteins. Mechanical force is not only a relevant quantity in cellular protein folding and function, but also a convenient parameter for biophysical folding studies. Optical tweezers offer precise control in the force range relevant for protein folding and unfolding, from which single-molecule kinetic and thermodynamic information about these processes can be extracted. In this review, we describe both physical principles and practical aspects of optical tweezers measurements and discuss recent advances in the use of this technique for the study of protein folding. In particular, we describe the characterization of folding energy landscapes at high resolution, studies of structurally complex multidomain proteins, folding in the presence of chaperones, and the ability to investigate real-time cotranslational folding of a polypeptide.
Asunto(s)
Escherichia coli/genética , Chaperonas Moleculares/genética , Pinzas Ópticas , Biosíntesis de Proteínas , Proteoma/química , Ribosomas/genética , Escherichia coli/metabolismo , Humanos , Cinética , Microscopía de Fuerza Atómica , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteoma/biosíntesis , Proteoma/genética , Proteostasis/genética , Ribosomas/metabolismo , Ribosomas/ultraestructura , TermodinámicaRESUMEN
DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.
Asunto(s)
ADN/genética , Edición Génica/métodos , Técnicas de Transferencia de Gen , Genes Sintéticos , Ingeniería Genética/métodos , Genoma , Sistemas CRISPR-Cas , ADN/química , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Poliovirus/genética , Poliovirus/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esferoplastos/genética , Esferoplastos/metabolismoRESUMEN
Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures.
Asunto(s)
Escherichia coli/genética , Chaperonas Moleculares/genética , Biosíntesis de Proteínas , Proteoma/química , Ribosomas/genética , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteoma/biosíntesis , Proteoma/genética , Proteostasis/genética , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patología , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.
Asunto(s)
Escherichia coli/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Ribosomas/genética , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Poli A/química , Poli A/genética , Poli A/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteolisis , Empalme del ARN , Estabilidad del ARN , Ribosomas/metabolismo , Ribosomas/ultraestructura , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.
Asunto(s)
Hemiterpenos/biosíntesis , Hevea/metabolismo , Látex/biosíntesis , Proteínas de Plantas/química , Goma/química , Transferasas/química , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hemiterpenos/química , Hemiterpenos/metabolismo , Hevea/química , Hevea/genética , Látex/química , Látex/metabolismo , Modelos Moleculares , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Goma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terpenos/química , Terpenos/metabolismo , Transferasas/genética , Transferasas/metabolismoRESUMEN
Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucolípidos/biosíntesis , Antígenos O/biosíntesis , Poliprenoles/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Ligasas de Carbono-Oxígeno/química , Ligasas de Carbono-Oxígeno/genética , Ligasas de Carbono-Oxígeno/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ácidos Teicoicos/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide, which acts as a barrier and contributes to antibiotic resistance. The systems that mediate phospholipid trafficking across the periplasm, such as MCE (Mammalian Cell Entry) transporters, have not been well characterized. Our ~3.5 Å cryo-EM structure of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Our results support a model in which LetB establishes a physical link between the two membranes and creates a hydrophobic pathway for the translocation of lipids across the periplasm.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/fisiología , Transporte Biológico , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Fosfolípidos/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.
Asunto(s)
Escherichia coli/metabolismo , Ingeniería Metabólica , Metanol/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Ciclo del Ácido Cítrico/genética , Variaciones en el Número de Copia de ADN , Evolución Molecular Dirigida , Escherichia coli/genética , Formaldehído/metabolismo , Glucólisis , Mutagénesis , Ribosamonofosfatos/metabolismoRESUMEN
Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.