RESUMEN
Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle1,2. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.
Asunto(s)
Amoníaco , Organismos Acuáticos , Archaea , Membrana Celular , Amoníaco/química , Amoníaco/metabolismo , Organismos Acuáticos/química , Organismos Acuáticos/metabolismo , Organismos Acuáticos/ultraestructura , Archaea/química , Archaea/metabolismo , Archaea/ultraestructura , Cationes/química , Cationes/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Oxidación-Reducción , Polisacáridos/metabolismo , Polisacáridos/químicaRESUMEN
Lipopolysaccharide (LPS) transport to the outer membrane (OM) is a crucial step in the biogenesis of microbial surface defenses. Although many features of the translocation mechanism have been elucidated, molecular details of LPS insertion via the LPS transport (Lpt) OM protein LptDE remain elusive. Here, we integrate native MS with hydrogen-deuterium exchange MS and molecular dynamics simulations to investigate the influence of substrate and peptide binding on the conformational dynamics of LptDE. Our data reveal that LPS induces opening of the LptD ß-taco domain, coupled with conformational changes on ß-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilizes the ß-taco, thereby preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both the ß-barrel and ß-taco domains of LptD, and suggest a means for developing antimicrobial therapeutics targeting this essential process in Gram-negative ESKAPE pathogens.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Lipopolisacáridos/metabolismo , Translocación Genética/genética , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Conformación de Carbohidratos , Farmacorresistencia Bacteriana/genética , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Espectrometría de Masas , Modelos Moleculares , Simulación de Dinámica MolecularRESUMEN
Chemotaxis signaling pathways enable bacteria to sense and respond to their chemical environment and, in some species, are critical for lifestyle processes such as biofilm formation and pathogenesis. The signal transduction underlying chemotaxis behavior is mediated by large, highly ordered protein complexes known as chemosensory arrays. For nearly two decades, cryo-electron tomography (cryoET) has been used to image chemosensory arrays, providing an increasingly detailed understanding of their structure and function. In this mini-review, we provide an overview of the use of cryoET to study chemosensory arrays, including imaging strategies, key results, and outstanding questions. We further discuss the application of molecular modeling and simulation techniques to complement structure determination efforts and provide insight into signaling mechanisms. We close the review with a brief outlook, highlighting promising future directions for the field.
Asunto(s)
Quimiotaxis , Escherichia coli , Quimiotaxis/fisiología , Escherichia coli/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/química , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Tomografía con Microscopio Electrónico , Histidina Quinasa , Transducción de Señal , Proteínas Bacterianas/metabolismoRESUMEN
Stimulation of Escherichia coli with acetate elevates the acetylation level of the chemotaxis response regulator CheY. This elevation, in an unknown mechanism, activates CheY to generate clockwise rotation. Here, using quantitative selective reaction monitoring mass spectrometry and high-resolution targeted mass spectrometry, we identified K91 and K109 as the major sites whose acetylation level in vivo increases in response to acetate. Employing single and multiple lysine replacements in CheY, we found that K91 and K109 are also the sites mainly responsible for acetate-dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K91 is nonmodified, as evidenced by an increased ability of CheY to generate clockwise rotation when K91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations, we show that K91 repression is manifested in the conformational dynamics of the ß4α4 loop, shifted toward an active state upon mutation. Removal of ß4α4 loop repression may represent a general activation mechanism in CheY, pertaining also to the canonical phosphorylation activation pathway as suggested by crystal structures of active and inactive CheY from Thermotoga maritima. By way of elimination, we further suggest that K109 acetylation is actively involved in generating clockwise rotation.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Acetatos/metabolismo , Acetilación , Quimiotaxis , Escherichia coli/química , Proteínas de Escherichia coli , Lisina/metabolismo , Espectrometría de Masas , Proteínas Quimiotácticas Aceptoras de Metilo , Simulación de Dinámica Molecular , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Thermotoga maritima/metabolismoRESUMEN
Certain inhibitor of apoptosis (IAP) family members are sentinel proteins that prevent untimely cell death by inhibiting caspases. Antagonists, including second mitochondria-derived activator of caspases (SMAC), regulate IAPs and drive cell death. Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP with dual E2 and E3 ubiquitin ligase activity, regulates programmed cell death through unknown mechanisms. We show that BIRC6 directly restricts executioner caspase-3 and -7 and ubiquitinates caspase-3, -7, and -9, working exclusively with noncanonical E1, UBA6. Notably, we show that SMAC suppresses both mechanisms. Cryo-electron microscopy structures of BIRC6 alone and in complex with SMAC reveal that BIRC6 is an antiparallel dimer juxtaposing the substrate-binding module against the catalytic domain. Furthermore, we discover that SMAC multisite binding to BIRC6 results in a subnanomolar affinity interaction, enabling SMAC to competitively displace caspases, thus antagonizing BIRC6 anticaspase function.
Asunto(s)
Apoptosis , Caspasa 3 , Caspasa 7 , Caspasa 9 , Proteínas Inhibidoras de la Apoptosis , Ubiquitina-Proteína Ligasas , Humanos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Caspasa 9/metabolismo , Microscopía por Crioelectrón , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Dominio Catalítico , Multimerización de ProteínaRESUMEN
IMPORTANCE: Bacterial chemotaxis is a ubiquitous behavior that enables cell movement toward or away from specific chemicals. It serves as an important model for understanding cell sensory signal transduction and motility. Characterization of the molecular mechanisms underlying chemotaxis is of fundamental interest and requires a high-resolution structural picture of the sensing machinery, the chemosensory array. In this study, we combine cryo-electron tomography and molecular simulation to present the complete structure of the core signaling unit, the basic building block of chemosensory arrays, from Escherichia coli. Our results provide new insight into previously poorly-resolved regions of the complex and offer a structural basis for designing new experiments to test mechanistic hypotheses.
Asunto(s)
Quimiotaxis , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/química , Proteínas Quimiotácticas Aceptoras de Metilo/química , Transducción de Señal , Proteínas de Escherichia coli/química , Proteínas Bacterianas/químicaRESUMEN
Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities.
Asunto(s)
Proteínas de la Membrana , Simulación de Dinámica Molecular , Proteínas de la Membrana/química , Microscopía por Crioelectrón , Lípidos de la Membrana , Conformación ProteicaRESUMEN
The Escherichia coli chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)-based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Quimiotaxis , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Histidina Quinasa/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/genéticaRESUMEN
Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high-resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 Å structure indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.
Asunto(s)
Bacterias , Microscopía por Crioelectrón , Rayos Ultravioleta , Virus , Bacterias/patogenicidad , Bacterias/efectos de la radiación , Quimiotaxis/efectos de la radiación , Vibrio cholerae/patogenicidad , Vibrio cholerae/efectos de la radiación , Virus/patogenicidad , Virus/efectos de la radiaciónRESUMEN
Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these protein-lipid interactions. Systematic comparative analysis requires tools that provide algorithms for objective assessment of such interactions. We introduce PyLipID, a Python package for the identification and characterization of specific lipid interactions and binding sites on membrane proteins from molecular dynamics simulations. PyLipID uses a community analysis approach for binding site detection, calculating lipid residence times for both the individual protein residues and the detected binding sites. To assist structural analysis, PyLipID produces representative bound lipid poses from simulation data, using a density-based scoring function. To estimate residue contacts robustly, PyLipID uses a dual-cutoff scheme to differentiate between lipid conformational rearrangements while bound from full dissociation events. In addition to the characterization of protein-lipid interactions, PyLipID is applicable to analysis of the interactions of membrane proteins with other ligands. By combining automated analysis, efficient algorithms, and open-source distribution, PyLipID facilitates the systematic analysis of lipid interactions from large simulation data sets of multiple species of membrane proteins.
Asunto(s)
Proteínas de la Membrana , Simulación de Dinámica Molecular , Sitios de Unión , Ligandos , Membrana Dobles de Lípidos/química , Lípidos , Proteínas de la Membrana/químicaRESUMEN
The Klebsiella jumbo myophage ÏKp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combine cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, microbiological and machine learning approaches to explore the capsid, tail, and tail fibers of ÏKp24. We determine the structure of the capsid and tail at 4.1 Å and 3.0 Å resolution. We observe the tail fibers are branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide ÏKp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Our study provides structural and functional insight into how ÏKp24 adapts to the variable surfaces of capsulated bacterial pathogens, which is useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.
Asunto(s)
Bacteriófagos , Microscopía por Crioelectrón , Klebsiella pneumoniae , Klebsiella , Cápside , Proteínas de la CápsideRESUMEN
Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.
Asunto(s)
Methylococcaceae , Oxigenasas , Cobre/química , Metano/metabolismo , Methylococcaceae/metabolismo , Minerales , Oxidación-Reducción , Oxigenasas/metabolismoRESUMEN
Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here, we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays appear to exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Histidina Quinasa/química , Proteínas Quimiotácticas Aceptoras de Metilo/química , Quimiotaxis/fisiología , Microscopía por Crioelectrón , Dimerización , Modelos MolecularesRESUMEN
Bacteria that colonize animals must overcome, or coexist, with the reactive oxygen species products of inflammation, a front-line defense of innate immunity. Among these is the neutrophilic oxidant bleach, hypochlorous acid (HOCl), a potent antimicrobial that plays a primary role in killing bacteria through nonspecific oxidation of proteins, lipids, and DNA. Here, we report that in response to increasing HOCl levels, Escherichia coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We identify the mechanism of DgcZ sensing of HOCl to be direct oxidation of its regulatory chemoreceptor zinc-binding (CZB) domain. Dissection of CZB signal transduction reveals that oxidation of the conserved zinc-binding cysteine controls CZB Zn2+ occupancy, which in turn regulates the catalysis of c-di-GMP by the associated GGDEF domain. We find DgcZ-dependent biofilm formation and HOCl sensing to be regulated in vivo by the conserved zinc-coordinating cysteine. Additionally, point mutants that mimic oxidized CZB states increase total biofilm. A survey of bacterial genomes reveals that many pathogenic bacteria that manipulate host inflammation as part of their colonization strategy possess CZB-regulated diguanylate cyclases and chemoreceptors. Our findings suggest that CZB domains are zinc-sensitive regulators that allow host-associated bacteria to perceive host inflammation through reactivity with HOCl. IMPORTANCE Immune cells are well equipped to eliminate invading bacteria, and one of their primary tools is the synthesis of bleach, hypochlorous acid (HOCl), the same chemical used as a household disinfectant. In this work, we present findings showing that many host-associated bacteria possess a bleach-sensing protein that allows them to adapt to the presence of this chemical in their environment. We find that the bacterium Escherichia coli responds to bleach by hunkering down and producing a sticky matrix known as biofilm, which helps it aggregate and adhere to surfaces. This behavior may play an important role in pathogenicity for E. coli and other bacteria, as it allows the bacteria to detect and adapt to the weapons of the host immune system.
Asunto(s)
Adhesión Bacteriana/genética , Biopelículas/crecimiento & desarrollo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Inflamación/genética , Transducción de Señal , Bacterias/clasificación , Bacterias/genética , Bacterias/inmunología , Bacterias/metabolismo , Adhesión Bacteriana/inmunología , Biopelículas/efectos de los fármacos , GMP Cíclico/genética , GMP Cíclico/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/inmunología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/inmunología , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Ácido Hipocloroso/farmacología , Inflamación/inmunologíaRESUMEN
To enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.
Asunto(s)
Tomografía con Microscopio Electrónico , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Conformación Molecular , Simulación de Dinámica Molecular , Proteínas Adaptadoras Transductoras de Señales , Quimiotaxis , Escherichia coliRESUMEN
Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Bacillus subtilis Ethanol and other short-chain alcohols are attractants for B. subtilis Two chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the signaling domain directly binds ethanol. We were further able to identify a single amino acid residue, Ala431, on the cytoplasmic signaling domain of McpB that, when mutated to serine, reduces taxis to alcohols. Molecular dynamics simulations suggest that the conversion of Ala431 to serine increases coiled-coil packing within the signaling domain, thereby reducing the ability of ethanol to bind between the helices of the signaling domain. In the case of HemAT, the myoglobin-like sensing domain binds ethanol, likely between the helices encapsulating the heme group. Aside from being sensed by an unconventional mechanism, ethanol also differs from many other chemoattractants because it is not metabolized by B. subtilis and is toxic. We propose that B. subtilis uses ethanol and other short-chain alcohols to locate prey, namely, alcohol-producing microorganisms.IMPORTANCE Ethanol is a chemoattractant for Bacillus subtilis even though it is not metabolized and inhibits growth. B. subtilis likely uses ethanol to find ethanol-fermenting microorganisms to utilize as prey. Two chemoreceptors sense ethanol: HemAT and McpB. HemAT's myoglobin-like sensing domain directly binds ethanol, but the heme group is not involved. McpB is a transmembrane receptor consisting of an extracellular sensing domain and a cytoplasmic signaling domain. While most attractants bind the extracellular sensing domain, we found that ethanol directly binds between intermonomer helices of the cytoplasmic signaling domain of McpB, using a mechanism akin to those identified in many mammalian ethanol-binding proteins. Our results indicate that the sensory repertoire of chemoreceptors extends beyond the sensing domain and can directly involve the signaling domain.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Quimiotaxis , Citoplasma/metabolismo , Etanol/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Células Quimiorreceptoras/metabolismo , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Transducción de SeñalRESUMEN
Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Quimiotácticas Aceptoras de Metilo/química , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Escherichia coli/genética , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestructura , Histidina Quinasa/química , Histidina Quinasa/genética , Histidina Quinasa/ultraestructura , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Proteínas Quimiotácticas Aceptoras de Metilo/ultraestructura , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructuraRESUMEN
Tsr, the serine chemoreceptor in Escherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCE In Escherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the E. coli serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.
Asunto(s)
Escherichia coli/enzimología , Proteínas Quimiotácticas Aceptoras de Metilo/química , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Multimerización de Proteína , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Escherichia coli/fisiología , Simulación de Dinámica Molecular , Conformación Proteica , Transducción de SeñalRESUMEN
Recent advances in cryo-electron microscopy (cryoEM) have dramatically improved the resolutions at which vitrified biological specimens can be studied, revealing new structural and mechanistic insights over a broad range of spatial scales. Bolstered by these advances, much effort has been directed toward the development of hybrid modeling methodologies for the construction and refinement of high-fidelity atomistic models from cryoEM data. In this brief review, we will survey the key elements of cryoEM-based hybrid modeling, providing an overview of available computational tools and strategies as well as several recent applications.
Asunto(s)
Microscopía por Crioelectrón/métodos , Modelos Moleculares , Biología Computacional , Simulación por Computador , Conformación ProteicaRESUMEN
The rise of the computer as a powerful tool for model building and refinement has revolutionized the field of structure determination for large biomolecular systems. Despite the wide availability of robust experimental methods capable of resolving structural details across a range of spatiotemporal resolutions, computational hybrid methods have the unique ability to integrate the diverse data from multimodal techniques such as X-ray crystallography and electron microscopy into consistent, fully atomistic structures. Here, commonly employed strategies for computational real-space structural refinement are reviewed, and their specific applications are illustrated for several large macromolecular complexes: ribosome, virus capsids, chemosensory array, and photosynthetic chromatophore. The increasingly important role of computational methods in large-scale structural refinement, along with current and future challenges, is discussed.