RESUMEN
The autosomal dominant monogenetic disease neurofibromatosis type 1 (NF1) affects approximately one in 3,000 individuals and is caused by mutations in the NF1 tumour suppressor gene, leading to dysfunction in the protein neurofibromin (Nf1)1,2. As a GTPase-activating protein, a key function of Nf1 is repression of the Ras oncogene signalling cascade. We determined the human Nf1 dimer structure at an overall resolution of 3.3 Å. The cryo-electron microscopy structure reveals domain organization and structural details of the Nf1 exon 23a splicing3 isoform 2 in a closed, self-inhibited, Zn-stabilized state and an open state. In the closed conformation, HEAT/ARM core domains shield the GTPase-activating protein-related domain (GRD) so that Ras binding is sterically inhibited. In a distinctly different, open conformation of one protomer, a large-scale movement of the GRD occurs, which is necessary to access Ras, whereas Sec14-PH reorients to allow interaction with the cellular membrane4. Zn incubation of Nf1 leads to reduced Ras-GAP activity with both protomers in the self-inhibited, closed conformation stabilized by a Zn binding site between the N-HEAT/ARM domain and the GRD-Sec14-PH linker. The transition between closed, self-inhibited states of Nf1 and open states provides guidance for targeted studies deciphering the complex molecular mechanism behind the widespread neurofibromatosis syndrome and Nf1 dysfunction in carcinogenesis.
Asunto(s)
Microscopía por Crioelectrón , Neurofibromina 2/química , Neurofibromina 2/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Empalme Alternativo , Sitios de Unión , Exones , Humanos , Modelos Moleculares , Neurofibromina 1/metabolismo , Neurofibromina 2/ultraestructura , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestructura , Multimerización de Proteína , Estabilidad Proteica , Zinc/metabolismoRESUMEN
Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) "domain". Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo-electron microscopy study of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of heteromeric vertebrate egg coat filaments identify a common sperm-binding region at the interface between subunits.
Asunto(s)
Polímeros/química , Uromodulina/química , Zona Pelúcida/química , Secuencia de Aminoácidos , Animales , Microscopía por Crioelectrón/métodos , Femenino , Humanos , Polimerizacion , Polímeros/metabolismo , Conformación Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Uromodulina/genética , Uromodulina/metabolismo , Zona Pelúcida/metabolismoRESUMEN
BACKGROUND: NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. RESULTS: We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that "plugs" the NorQ ring pore to the VWA domain of NorD with a protruding "finger" inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples. CONCLUSIONS: Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.
Asunto(s)
Proteínas AAA , Paracoccus denitrificans , Chaperonas Moleculares , Noretindrona , Relación Estructura-ActividadRESUMEN
The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1-ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Endopeptidasa Clp/metabolismo , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis , Oligopéptidos/farmacología , Proteínas Bacterianas/química , Endopeptidasa Clp/química , Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/metabolismo , Péptido Hidrolasas/metabolismo , Péptidos/metabolismoRESUMEN
Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson's disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.
Asunto(s)
Amiloide/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Enfermedad de Parkinson/metabolismo , Amiloide/química , Amiloide/ultraestructura , Tomografía con Microscopio Electrónico , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas del Choque Térmico HSP110/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Técnicas In Vitro , Cinética , Chaperonas Moleculares/metabolismo , Enfermedad de Parkinson/etiología , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Multimerización de Proteína , Solubilidad , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo-electron microscopy (cryo-EM) structure of the heterodimeric DP1-DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1-DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.
Asunto(s)
Proteínas de Unión al ADN/ultraestructura , Factor de Transcripción DP1/ultraestructura , Factores de Transcripción/ultraestructura , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , Dominio Catalítico , Microscopía por Crioelectrón/métodos , ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/ultraestructura , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , Dominios Proteicos/genética , Subunidades de Proteína/metabolismo , Pyrococcus abyssi/metabolismo , Pyrococcus abyssi/ultraestructura , Factor de Transcripción DP1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Cryo-electron microscopy (cryo-EM) is a structural molecular and cellular biology technique that has experienced major advances in recent years. Technological developments in image recording as well as in processing software make it possible to obtain three-dimensional reconstructions of macromolecular assemblies at near-atomic resolution that were formerly obtained only by X-ray crystallography or NMR spectroscopy. In parallel, cryo-electron tomography has also benefitted from these technological advances, so that visualization of irregular complexes, organelles or whole cells with their molecular machines in situ has reached subnanometre resolution. Cryo-EM can therefore address a broad range of biological questions. The aim of this review is to provide a brief overview of the principles and current state of the cryo-EM field.
Asunto(s)
Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Sustancias Macromoleculares/ultraestructura , Microscopía por Crioelectrón/instrumentación , Cristalografía por Rayos X , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Sustancias Macromoleculares/química , Modelos Moleculares , Conformación Proteica , Programas InformáticosRESUMEN
Cryogenic electron microscopy (cryo-EM) has become in the past 10 years one of the major tools for the structure determination of proteins. Nowadays, the structure prediction field is experiencing the same revolution and, using AlphaFold2, it is possible to have high-confidence atomic models for virtually any polypeptide chain, smaller than 4000 amino acids, in a simple click. Even in a scenario where all polypeptide chain folding were to be known, cryo-EM retains specific characteristics that make it a unique tool for the structure determination of macromolecular complexes. Using cryo-EM, it is possible to obtain near-atomic structures of large and flexible mega-complexes, describe conformational panoramas, and potentially develop a structural proteomic approach from fully ex vivo specimens.
Asunto(s)
Proteínas , Proteómica , Microscopía por Crioelectrón , Sustancias Macromoleculares/química , PéptidosRESUMEN
Cryogenic electron microscopy (cryo-EM) is constantly developing and growing as a major technique for structure determination of protein complexes. Here, we detail the first steps of any cryo-EM project: specimen preparation and data collection. Step by step, a list of material needed is provided and the sequence of actions to carry out is given. We hope that these protocols will be useful to all people getting started with cryo-EM.
Asunto(s)
Proteínas , Manejo de Especímenes , Humanos , Microscopía por Crioelectrón/métodos , Manejo de Especímenes/métodosRESUMEN
Glycoprotein 2 (GP2) and uromodulin (UMOD) filaments protect against gastrointestinal and urinary tract infections by acting as decoys for bacterial fimbrial lectin FimH. By combining AlphaFold2 predictions with X-ray crystallography and cryo-EM, we show that these proteins contain a bipartite decoy module whose new fold presents the high-mannose glycan recognized by FimH. The structure rationalizes UMOD mutations associated with kidney diseases and visualizes a key epitope implicated in cast nephropathy.
Asunto(s)
Adhesinas Bacterianas , Fimbrias Bacterianas , Adhesinas Bacterianas/genética , Cristalografía por Rayos X , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/química , Fimbrias Bacterianas/metabolismo , Proteínas Ligadas a GPI , Humanos , Manosa/análisis , Uromodulina/análisis , Uromodulina/química , Uromodulina/metabolismoRESUMEN
Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here Single Particle Automated Raman Trapping Analysis (SPARTA) is combined with small-angle X-ray and neutron scattering (SAXS/SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP-phospholipase D (PLD) interactions. This analysis demonstrates that PLD, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate is an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated.
Asunto(s)
Lípidos , Nanopartículas , Lípidos/química , Liposomas , Nanopartículas/química , Fosfolipasas , ARN Interferente Pequeño/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate carbon hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal concentrations. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED.
RESUMEN
Scipion is a modular image-processing framework that integrates several software packages under a unified interface while taking care of file formats and conversions. Here, new developments and capabilities of the Scipion plugin for the widely used RELION software package are presented and illustrated with an image-processing pipeline for published data. The user interfaces of Scipion and RELION are compared and the key differences are highlighted, allowing this manuscript to be used as a guide for both new and experienced users of this software. Different on-the-fly image-processing options are also discussed, demonstrating the flexibility of the Scipion framework.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Modelos Moleculares , Programas InformáticosRESUMEN
Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the ß1-ß2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.
Asunto(s)
Cianobacterias/metabolismo , Canales Iónicos Activados por Ligandos/química , Proteínas Bacterianas/química , Microscopía por Crioelectrón , Cianobacterias/química , Concentración de Iones de Hidrógeno , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Dominios ProteicosRESUMEN
IscS and IscU, the two central protein components of the iron sulfur cluster assembly machinery, form a complex that is still relatively poorly characterized. In an attempt to standardize the purification of these proteins for structural studies we have developed a protocol to produce them individually in high concentration and purity. We show that IscS is a rather robust protein as long as it is produced in a PLP loaded form and that this co-factor is essential for fold stability and enzyme activity. In contrast to previous evidence, we also propose that, in contrast with previous evidence, IscU is a thermodynamically stable protein with a well defined fold but, when produced in isolation, is a 'complex-orphan protein' that is prone to unfolding if not stabilised by a co-factor or a protein partner. Our work will facilitate further structural and functional studies of these proteins and eventually lead to a better understanding of the whole machinery.
Asunto(s)
Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas/metabolismo , Compuestos de Sulfonilurea/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/aislamiento & purificación , Cisteína/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/aislamiento & purificación , Unión Proteica , Compuestos de Sulfonilurea/química , Compuestos de Sulfonilurea/aislamiento & purificaciónRESUMEN
Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Archaea , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Clonación Molecular , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/química , ADN Polimerasa Dirigida por ADN/genética , Eucariontes , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Pyrococcus abyssi/genética , Pyrococcus abyssi/metabolismo , Proteínas Recombinantes de FusiónRESUMEN
Bacteriophage PR772, a member of the Tectiviridae family, has a 70 nm diameter icosahedral protein capsid that encapsulates a lipid membrane, dsDNA, and various internal proteins. An icosahedrally averaged CryoEM reconstruction of the wild-type virion and a localized reconstruction of the vertex region reveal the composition and the structure of the vertex complex along with new protein conformations that play a vital role in maintaining the capsid architecture of the virion. The overall resolution of the virion is 2.75 Å, while the resolution of the protein capsid is 2.3 Å. The conventional penta-symmetron formed by the capsomeres is replaced by a large vertex complex in the pseudo T = 25 capsid. All the vertices contain the host-recognition protein, P5; two of these vertices show the presence of the receptor-binding protein, P2. The 3D structure of the vertex complex shows interactions with the viral membrane, indicating a possible mechanism for viral infection.
Asunto(s)
Bacteriófagos/ultraestructura , Cápside/ultraestructura , Microscopía por Crioelectrón , Tectiviridae/ultraestructura , Proteínas de la Cápside/ultraestructura , Procesamiento de Imagen Asistido por Computador , Conformación ProteicaRESUMEN
Respiratory chain complexes execute energy conversion by connecting electron transport with proton translocation over the inner mitochondrial membrane to fuel ATP synthesis. Notably, these complexes form multi-enzyme assemblies known as respiratory supercomplexes. Here we used single-particle cryo-EM to determine the structures of the yeast mitochondrial respiratory supercomplexes III2IV and III2IV2, at 3.2-Å and 3.5-Å resolutions, respectively. We revealed the overall architecture of the supercomplex, which deviates from the previously determined assemblies in mammals; obtained a near-atomic structure of the yeast complex IV; and identified the protein-protein and protein-lipid interactions implicated in supercomplex formation. Take together, our results demonstrate convergent evolution of supercomplexes in mitochondria that, while building similar assemblies, results in substantially different arrangements and structural solutions to support energy conversion.
Asunto(s)
Microscopía por Crioelectrón/métodos , Animales , Transporte de Electrón/fisiología , Humanos , Metabolismo de los Lípidos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructuraRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
RESUMEN
ATP-driven bacterial AAA+ proteases have been recognized as drug targets. They possess an AAA+ protein (e.g., ClpC), which threads substrate proteins into an associated peptidase (e.g., ClpP). ATPase activity and substrate selection of AAA+ proteins are regulated by adapter proteins that bind to regulatory domains, such as the N-terminal domain (NTD). The antibacterial peptide Cyclomarin A (CymA) kills Mycobacterium tuberculosis cells by binding to the NTD of ClpC. How CymA affects ClpC function is unknown. Here, we reveal the mechanism of CymA-induced toxicity. We engineered a CymA-sensitized ClpC chimera and show that CymA activates ATPase and proteolytic activities. CymA mimics adapter binding and enables autonomous protein degradation by ClpC/ClpP with relaxed substrate selectivity. We reconstitute CymA toxicity in E. coli cells expressing engineered ClpC and ClpP, demonstrating that gain of uncontrolled proteolytic activity causes cell death. This validates drug-induced overriding of AAA+ protease activity control as effective antibacterial strategy.