RESUMEN
Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a ß-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the ß-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Chaperonas Moleculares , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/química , Transporte de Proteínas , Pliegue de Proteína , Bacterias Gramnegativas/metabolismo , Bacterias Gramnegativas/genética , Membrana Externa Bacteriana/metabolismo , Modelos Moleculares , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Canales de Translocación SEC/metabolismo , Canales de Translocación SEC/genética , Canales de Translocación SEC/química , Periplasma/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
Cell shape matters across the kingdoms of life, and cells have the remarkable capacity to define and maintain specific shapes and sizes. But how are the shapes of micron-sized cells determined from the coordinated activities of nanometer-sized proteins? Here, we review general principles that have surfaced through the study of rod-shaped bacterial growth. Imaging approaches have revealed that polymers of the actin homolog MreB play a central role. MreB both senses and changes cell shape, thereby generating a self-organizing feedback system for shape maintenance. At the molecular level, structural and computational studies indicate that MreB filaments exhibit tunable mechanical properties that explain their preference for certain geometries and orientations along the cylindrical cell body. We illustrate the regulatory landscape of rod-shape formation and the connectivity between cell shape, cell growth, and other aspects of cell physiology. These discoveries provide a framework for future investigations into the architecture and construction of microbes.
Asunto(s)
Membrana Celular/genética , Pared Celular/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Membrana Celular/metabolismo , Pared Celular/metabolismo , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending â¼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for toxin transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane. VIDEO ABSTRACT.
Asunto(s)
Antibiosis , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Sistemas de Secreción Tipo V/metabolismo , Extensiones de la Superficie Celular/metabolismo , Extensiones de la Superficie Celular/ultraestructura , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de la Membrana/química , Dominios Proteicos , Receptores de Superficie Celular/metabolismoRESUMEN
mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Mensajero/metabolismo , Algoritmos , Sitios de Unión , Sistema Libre de Células , Cartilla de ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Entropía , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Pliegue del ARN , ARN Mensajero/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Ribosomas/química , Ribosomas/metabolismo , Regiones no TraducidasRESUMEN
Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.
Asunto(s)
Colesterol/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Microscopía por Crioelectrón , Dimerización , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Ratones , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Receptor Patched-1/química , Receptor Patched-1/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Transducción de SeñalRESUMEN
NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Factores de Elongación de Péptidos/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Dominio Catalítico , Microscopía por Crioelectrón , ADN/química , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Conformación de Ácido Nucleico , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Operón de ARNr/genéticaRESUMEN
Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here, we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes IscR to repress expression of a TMAO-responsive signaling system, allowing stochastic effects to have a strong effect on the output of the system and resulting in heterogeneous expression of the TMAO reduction machinery. This work reveals a mechanism through which cells regulate molecular noise to enhance fitness.
Asunto(s)
Escherichia coli/metabolismo , Transducción de Señal , Aerobiosis , Anaerobiosis , Secuencia de Bases , Sitios de Unión , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metilaminas/metabolismo , Metilaminas/farmacología , Oxígeno/metabolismo , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Fosfotransferasas/química , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de la Membrana/química , Membrana Celular/química , Cristalografía por Rayos X , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/químicaRESUMEN
Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a trans-esterase activity, which is responsible for nicking the DNA strand to be transferred and for covalent attachment to the resulting 5'-phosphate end, and a helicase activity, which is responsible for unwinding the DNA while it is being transported to a recipient cell. Here we show that these two activities are carried out by two conformers that can both load simultaneously on the origin of transfer DNA. We solve the structure of one of these conformers by cryo electron microscopy to near-atomic resolution, elucidating the molecular basis of helicase function by relaxases and revealing insights into the mechanistic events taking place in the cell prior to substrate transport during conjugation.
Asunto(s)
Conjugación Genética , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructura , Escherichia coli/genética , Microscopía por Crioelectrón , ADN Helicasas/química , ADN Bacteriano/química , ADN Bacteriano/ultraestructura , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos MolecularesRESUMEN
During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only â¼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging.
Asunto(s)
Proteínas de Escherichia coli , Factores de Transcripción , Factores de Transcripción/genética , Factores de Transcripción/química , Transcripción Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Factores de Elongación de Péptidos/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
In this issue of Molecular Cell, Roeselová et al.1 provide insights into co-translational folding of a multidomain protein in bacteria, revealing how the chaperones Trigger Factor, DnaJ, and DnaK work together to facilitate the folding of nascent chains.
Asunto(s)
Proteínas de Escherichia coli , Proteínas del Choque Térmico HSP40 , Chaperonas Moleculares , Pliegue de Proteína , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP40/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Biosíntesis de Proteínas , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/químicaRESUMEN
Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico , Biosíntesis de Proteínas , Pliegue de Proteína , Ribosomas , Ribosomas/metabolismo , Ribosomas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP40/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Unión Proteica , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Modelos Moleculares , Conformación Proteica , Isomerasa de PeptidilprolilRESUMEN
The determination of the crystal structures of small-molecule transporters has shed light on the conformational changes that take place during structural isomerization from outward- to inward-facing states. Rather than using a simple rocking movement of two bundles around a central substrate-binding site, it has become clear that even the most simplistic transporters utilize rearrangements of nonrigid bodies. In the most dramatic cases, one bundle is fixed while the other, structurally divergent, bundle carries the substrate some 18 Å across the membrane, which in this review is termed an elevator alternating-access mechanism. Here, we compare and contrast rocker-switch, rocking-bundle, and elevator alternating-access mechanisms to highlight shared features and novel refinements to the basic alternating-access model.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Membrana/química , Simulación de Dinámica Molecular , Transporte Biológico , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Expresión Génica , Cinética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
Recent developments indicate that macrodomains, an ancient and diverse protein domain family, are key players in the recognition, interpretation, and turnover of ADP-ribose (ADPr) signaling. Crucial to this is the ability of macrodomains to recognize ADPr either directly, in the form of a metabolic derivative, or as a modification covalently bound to proteins. Thus, macrodomains regulate a wide variety of cellular and organismal processes, including DNA damage repair, signal transduction, and immune response. Their importance is further indicated by the fact that dysregulation or mutation of a macrodomain is associated with several diseases, including cancer, developmental defects, and neurodegeneration. In this review, we summarize the current insights into macrodomain evolution and how this evolution influenced their structural and functional diversification. We highlight some aspects of macrodomain roles in pathobiology as well as their emerging potential as therapeutic targets.
Asunto(s)
Reparación del ADN , Proteínas de Escherichia coli/química , Neoplasias/enzimología , Poli(ADP-Ribosa) Polimerasas/química , Procesamiento Proteico-Postraduccional , Proteínas Represoras/química , Virosis/enzimología , Adenosina Difosfato Ribosa/química , Adenosina Difosfato Ribosa/metabolismo , Animales , Daño del ADN , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Familia de Multigenes , Neoplasias/química , Neoplasias/genética , Neoplasias/patología , Filogenia , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Dominios Proteicos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Homología Estructural de Proteína , Virosis/genética , Virosis/patología , Virosis/virologíaRESUMEN
Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod's remarkable mechanical properties and illuminates its role in pilus secretion.
Asunto(s)
Proteínas de Escherichia coli/química , Fimbrias Bacterianas/química , Escherichia coli Uropatógena/metabolismo , Microscopía por Crioelectrón , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Escherichia coli Uropatógena/citologíaRESUMEN
It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client's affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Periplasmáticas/metabolismo , Pliegue de Proteína , Proteínas Portadoras/metabolismo , Entropía , Interacciones Hidrofóbicas e Hidrofílicas , Periplasma/química , Electricidad EstáticaRESUMEN
Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F "sex" pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/fisiología , Factor F/química , Fimbrias Bacterianas/química , Modelos Moleculares , Fosfolípidos/química , Sitios de Ligazón Microbiológica/genética , Microscopía por Crioelectrón , Proteínas de Escherichia coli/metabolismo , Factor F/genética , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Lípidos/química , Mutación , Fosfolípidos/metabolismo , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Sistemas de Secreción Tipo V/química , Sistemas de Secreción Tipo V/metabolismoRESUMEN
Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3' overhang segments. The PAM-complementary sequence in the 3' overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Proteínas Asociadas a CRISPR/química , Cristalografía por Rayos X , Endodesoxirribonucleasas/química , Escherichia coli/genética , Escherichia coli/inmunología , Proteínas de Escherichia coli/química , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.