RESUMEN
Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.
Asunto(s)
Biología , Biología Computacional , Sustancias Macromoleculares/químicaRESUMEN
Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.
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Gametogénesis , Mitocondrias , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aldehído Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Coenzima A Ligasas/metabolismo , Microscopía por Crioelectrón , Citoplasma/metabolismo , Tomografía con Microscopio Electrónico , Meiosis , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Esporas Fúngicas/metabolismo , Modelos Moleculares , Estructura Cuaternaria de ProteínaRESUMEN
Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.
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Glicoproteínas de la Zona Pelúcida , Humanos , Masculino , Semen , Espermatozoides/química , Espermatozoides/metabolismo , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona Pelúcida/química , Glicoproteínas de la Zona Pelúcida/metabolismo , Óvulo/química , Óvulo/metabolismo , FemeninoRESUMEN
The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.
Asunto(s)
Receptor Nicotínico de Acetilcolina alfa 7 , Humanos , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/ultraestructura , Sitios de Unión , Microscopía por Crioelectrón , Inflamación/tratamiento farmacológico , Transducción de Señal , Regulación AlostéricaRESUMEN
The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.
RESUMEN
Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.
Asunto(s)
Ascomicetos , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Elementos Transponibles de ADN , Técnicas de Sustitución del Gen , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/ultraestructura , Microscopía por Crioelectrón , Ascomicetos/química , Ascomicetos/metabolismo , Ascomicetos/ultraestructuraRESUMEN
Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.
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Amiloide , Polipéptido Amiloide de los Islotes Pancreáticos , Humanos , Amiloide/química , Microscopía por Crioelectrón , Polipéptido Amiloide de los Islotes Pancreáticos/química , Proteínas AmiloidogénicasRESUMEN
Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.
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Virus de la Parotiditis , Infección Persistente , Humanos , Virus de la Parotiditis/fisiología , Nucleocápside , Fosfoproteínas/metabolismo , Replicación ViralRESUMEN
Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.
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Movimiento Celular , Glipicanos/química , Receptores de Netrina/química , Animales , Glipicanos/metabolismo , Humanos , Ratones , Proteínas Mutantes , Receptores de Netrina/metabolismo , Receptores de Superficie Celular/metabolismo , Anticuerpos de Dominio Único , TrombospondinasRESUMEN
Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rß1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rß1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rß2/IL-12Rß1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rß1 directly engages the common p40 subunit. We targeted the shared IL-12Rß1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.
Asunto(s)
Interleucina-12/química , Interleucina-12/metabolismo , Células Asesinas Naturales/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos/inmunología , Femenino , Células HEK293 , Humanos , Inmunidad , Interleucina-12/agonistas , Subunidad p40 de la Interleucina-12/química , Subunidad p40 de la Interleucina-12/metabolismo , Ratones Endogámicos C57BL , Modelos Moleculares , Neoplasias/inmunología , Neoplasias/patología , Estructura Cuaternaria de Proteína , Receptores de Interleucina/ultraestructura , Receptores de Interleucina-12/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
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.
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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
Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Secuencia de Aminoácidos , Animales , Disulfuros/metabolismo , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Transporte de Proteínas , Receptores Acoplados a Proteínas G/aislamiento & purificación , Receptores Acoplados a Proteínas G/ultraestructura , Homología Estructural de Proteína , Relación Estructura-Actividad , Proteínas Wnt/química , Proteínas Wnt/aislamiento & purificación , Proteínas Wnt/ultraestructuraRESUMEN
Both transcription and three-dimensional (3D) architecture of the mammalian genome play critical roles in neurodevelopment and its disorders. However, 3D genome structures of single brain cells have not been solved; little is known about the dynamics of single-cell transcriptome and 3D genome after birth. Here, we generated a transcriptome (3,517 cells) and 3D genome (3,646 cells) atlas of the developing mouse cortex and hippocampus by using our high-resolution multiple annealing and looping-based amplification cycles for digital transcriptomics (MALBAC-DT) and diploid chromatin conformation capture (Dip-C) methods and developing multi-omic analysis pipelines. In adults, 3D genome "structure types" delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first post-natal month. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, we examine allele-specific structure of imprinted genes, revealing local and chromosome (chr)-wide differences. These findings uncover an unknown dimension of neurodevelopment.
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Encéfalo/crecimiento & desarrollo , Genoma , Sensación/genética , Transcripción Genética , Alelos , Animales , Animales Recién Nacidos , Linaje de la Célula/genética , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Sitios Genéticos , Impresión Genómica , Ratones , Familia de Multigenes , Neuroglía/metabolismo , Neuronas/metabolismo , Transcriptoma/genética , Corteza Visual/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.
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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
Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Alucinógenos/química , Receptor de Serotonina 5-HT2A/química , Receptor de Serotonina 5-HT2A/metabolismo , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Expresión Génica , Células HEK293 , Alucinógenos/farmacología , Alucinógenos/uso terapéutico , Humanos , Ligandos , Dietilamida del Ácido Lisérgico/química , Dietilamida del Ácido Lisérgico/farmacología , Metiotepina/química , Metiotepina/metabolismo , Modelos Químicos , Mutación , Conformación Proteica en Hélice alfa , Receptor de Serotonina 5-HT2A/genética , Proteínas Recombinantes , Serotonina/metabolismo , SpodopteraRESUMEN
Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.
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Proteínas de la Membrana Bacteriana Externa/ultraestructura , Caulobacter crescentus/metabolismo , Glicoproteínas de Membrana/ultraestructura , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Caulobacter crescentus/ultraestructura , Microscopía por Crioelectrón/métodos , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/metabolismo , Tomografía/métodosRESUMEN
Tau aggregation into insoluble filaments is the defining pathological hallmark of tauopathies. However, it is not known what controls the formation and templated seeding of strain-specific structures associated with individual tauopathies. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of tau filaments from corticobasal degeneration (CBD) human brain tissue. Cryo-EM and mass spectrometry of tau filaments from CBD reveal that this conformer is heavily decorated with posttranslational modifications (PTMs), enabling us to map PTMs directly onto the structures. By comparing the structures and PTMs of tau filaments from CBD and Alzheimer's disease, it is found that ubiquitination of tau can mediate inter-protofilament interfaces. We propose a structure-based model in which cross-talk between PTMs influences tau filament structure, contributing to the structural diversity of tauopathy strains. Our approach establishes a framework for further elucidating the relationship between the structures of polymorphic fibrils, including their PTMs, and neurodegenerative disease.
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Procesamiento Proteico-Postraduccional , Tauopatías/metabolismo , Proteínas tau/química , Anciano , Microscopía por Crioelectrón , Femenino , Humanos , Masculino , Persona de Mediana Edad , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Tauopatías/patología , Proteínas tau/metabolismoRESUMEN
Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.
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Proteínas Bacterianas/ultraestructura , Flagelos/ultraestructura , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón/métodos , Flagelos/metabolismo , Conformación Proteica , TorqueRESUMEN
Lysosomal cholesterol egress requires two proteins, NPC1 and NPC2, whose defects are responsible for Niemann-Pick disease type C (NPC). Here, we present systematic structural characterizations that reveal the molecular basis for low-pH-dependent cholesterol delivery from NPC2 to the transmembrane (TM) domain of NPC1. At pH 8.0, similar structures of NPC1 were obtained in nanodiscs and in detergent at resolutions of 3.6 Å and 3.0 Å, respectively. A tunnel connecting the N-terminal domain (NTD) and the transmembrane sterol-sensing domain (SSD) was unveiled. At pH 5.5, the NTD exhibits two conformations, suggesting the motion for cholesterol delivery to the tunnel. A putative cholesterol molecule is found at the membrane boundary of the tunnel, and TM2 moves toward formation of a surface pocket on the SSD. Finally, the structure of the NPC1-NPC2 complex at 4.0 Å resolution was obtained at pH 5.5, elucidating the molecular basis for cholesterol handoff from NPC2 to NPC1(NTD).
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Colesterol/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Nanopartículas/química , Nanopartículas/ultraestructura , Proteína Niemann-Pick C1 , Dominios Proteicos , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
Integrin αvß8 binds with exquisite specificity to latent transforming growth factor-ß (L-TGF-ß). This binding is essential for activating L-TGF-ß presented by a variety of cell types. Inhibiting αvß8-mediated TGF-ß activation blocks immunosuppressive regulatory T cell differentiation, which is a potential therapeutic strategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvß8 ectodomain and its intact natural ligand, L-TGF-ß, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvß8 binding specificity and TGF-ß activation. Our studies reveal a mechanism of TGF-ß activation where mature TGF-ß signals within the confines of L-TGF-ß and the release and diffusion of TGF-ß are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvß8-mediated L-TGF-ß activation.