RESUMEN
Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.
Asunto(s)
Enfermedad , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Ensamble y Desensamble de Cromatina , Microscopía por Crioelectrón , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enfermedad/genética , Humanos , Mutación Missense/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología Estructural de Proteína , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (â¼5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to â¼4 Å or ATP plus Rbin-1, a chemical inhibitor, at â¼8 Å resolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an â¼20 nm long structured linker and a flexible â¼500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/química , Simulación de Dinámica Molecular , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimología , Secuencias de Aminoácidos , Animales , Sitios de Unión , Microscopía por Crioelectrón , Biogénesis de Organelos , Unión Proteica , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Células Sf9 , SpodopteraRESUMEN
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
Bacteria use rapid contraction of a long sheath of the type VI secretion system (T6SS) to deliver effectors into a target cell. Here, we present an atomic-resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four ß strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes.
Asunto(s)
Proteínas Bacterianas/química , Sistemas de Secreción Bacterianos , Vibrio cholerae/metabolismo , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Vibrio cholerae/química , Vibrio cholerae/citología , Vibrio cholerae/ultraestructuraRESUMEN
Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.
Asunto(s)
Antibiosis , Proteínas Bacterianas , Toxinas Bacterianas , Streptomyces , Antibacterianos/biosíntesis , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antibiosis/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proteínas Bacterianas/ultraestructura , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Microscopía por Crioelectrón , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Lectinas/ultraestructura , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Streptomyces/química , Streptomyces/efectos de los fármacos , Streptomyces/genética , Streptomyces/crecimiento & desarrollo , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces griseus/efectos de los fármacos , Streptomyces griseus/genética , Streptomyces griseus/crecimiento & desarrollo , Streptomyces griseus/metabolismoRESUMEN
There has been considerable recent progress in designing new proteins using deep-learning methods1-9. Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.
Asunto(s)
Aprendizaje Profundo , Proteínas , Dominio Catalítico , Microscopía por Crioelectrón , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/ultraestructura , Unión Proteica , Proteínas/química , Proteínas/metabolismo , Proteínas/ultraestructuraRESUMEN
Protein-RNA and protein-DNA complexes play critical roles in biology. Despite considerable recent advances in protein structure prediction, the prediction of the structures of protein-nucleic acid complexes without homology to known complexes is a largely unsolved problem. Here we extend the RoseTTAFold machine learning protein-structure-prediction approach to additionally predict nucleic acid and protein-nucleic acid complexes. We develop a single trained network, RoseTTAFoldNA, that rapidly produces three-dimensional structure models with confidence estimates for protein-DNA and protein-RNA complexes. Here we show that confident predictions have considerably higher accuracy than current state-of-the-art methods. RoseTTAFoldNA should be broadly useful for modeling the structure of naturally occurring protein-nucleic acid complexes, and for designing sequence-specific RNA and DNA-binding proteins.
Asunto(s)
Ácidos Nucleicos , ARN/química , Proteínas de Unión al ADN/química , ADN/químicaRESUMEN
The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein-nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , Microscopía por Crioelectrón/métodos , Ligandos , SARS-CoV-2 , COVID-19/virología , Escherichia coli , beta-Galactosidasa/química , beta-Galactosidasa/metabolismo , Conformación Proteica , Reproducibilidad de los ResultadosRESUMEN
There has been considerable recent progress in protein structure prediction using deep neural networks to predict inter-residue distances from amino acid sequences1-3. Here we investigate whether the information captured by such networks is sufficiently rich to generate new folded proteins with sequences unrelated to those of the naturally occurring proteins used in training the models. We generate random amino acid sequences, and input them into the trRosetta structure prediction network to predict starting residue-residue distance maps, which, as expected, are quite featureless. We then carry out Monte Carlo sampling in amino acid sequence space, optimizing the contrast (Kullback-Leibler divergence) between the inter-residue distance distributions predicted by the network and background distributions averaged over all proteins. Optimization from different random starting points resulted in novel proteins spanning a wide range of sequences and predicted structures. We obtained synthetic genes encoding 129 of the network-'hallucinated' sequences, and expressed and purified the proteins in Escherichia coli; 27 of the proteins yielded monodisperse species with circular dichroism spectra consistent with the hallucinated structures. We determined the three-dimensional structures of three of the hallucinated proteins, two by X-ray crystallography and one by NMR, and these closely matched the hallucinated models. Thus, deep networks trained to predict native protein structures from their sequences can be inverted to design new proteins, and such networks and methods should contribute alongside traditional physics-based models to the de novo design of proteins with new functions.
Asunto(s)
Redes Neurales de la Computación , Proteínas , Secuencia de Aminoácidos , Cristalografía por Rayos X , Alucinaciones , Humanos , Conformación Proteica , Proteínas/química , Proteínas/genéticaRESUMEN
Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
Asunto(s)
Simulación por Computador , Genes Sintéticos/genética , Canales Iónicos/química , Canales Iónicos/genética , Modelos Moleculares , Biología Sintética , Línea Celular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Conductividad Eléctrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canales Iónicos/metabolismo , Transporte Iónico , Liposomas/metabolismo , Técnicas de Placa-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Solubilidad , Agua/químicaRESUMEN
Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
Asunto(s)
Cloruros/metabolismo , Nucleótidos/metabolismo , Potasio/metabolismo , Simportadores/metabolismo , Animales , Línea Celular , Tamaño de la Célula , Humanos , Fosforilación/fisiología , Células Sf9 , Transducción de Señal/fisiología , Cotransportadores de K ClRESUMEN
Carbohydrates and glycoproteins modulate key biological functions. However, experimental structure determination of sugar polymers is notoriously difficult. Computational approaches can aid in carbohydrate structure prediction, structure determination, and design. In this work, we developed a glycan-modeling algorithm, GlycanTreeModeler, that computationally builds glycans layer-by-layer, using adaptive kernel density estimates (KDE) of common glycan conformations derived from data in the Protein Data Bank (PDB) and from quantum mechanics (QM) calculations. GlycanTreeModeler was benchmarked on a test set of glycan structures of varying lengths, or "trees". Structures predicted by GlycanTreeModeler agreed with native structures at high accuracy for both de novo modeling and experimental density-guided building. We employed these tools to design de novo glycan trees into a protein nanoparticle vaccine to shield regions of the scaffold from antibody recognition, and experimentally verified shielding. This work will inform glycoprotein model prediction, glycan masking, and further aid computational methods in experimental structure determination and refinement.
Asunto(s)
Algoritmos , Biología Computacional , Glicoproteínas , Modelos Moleculares , Polisacáridos , Polisacáridos/química , Biología Computacional/métodos , Glicoproteínas/química , Bases de Datos de Proteínas , Programas Informáticos , Conformación de CarbohidratosRESUMEN
Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1-8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10-12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10-12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11-14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.
Asunto(s)
Bacteriófago phi 6/química , Bacteriófago phi 6/ultraestructura , Microscopía por Crioelectrón , Empaquetamiento del ADN , Cristales Líquidos , Conformación de Ácido Nucleico , ARN Bicatenario/ultraestructura , ARN Viral/ultraestructura , Bacteriófago phi 6/genética , Genoma Viral , Modelos Moleculares , ARN Bicatenario/química , ARN Viral/química , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
Specificity of interactions between two DNA strands, or between protein and DNA, is often achieved by varying bases or side chains coming off the DNA or protein backbone-for example, the bases participating in Watson-Crick pairing in the double helix, or the side chains contacting DNA in TALEN-DNA complexes. By contrast, specificity of protein-protein interactions usually involves backbone shape complementarity1, which is less modular and hence harder to generalize. Coiled-coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions2) limits the number of orthogonal pairs that can be created simply by varying side-chain interactions3,4. Here we show that protein-protein interaction specificity can be achieved using extensive and modular side-chain hydrogen-bond networks. We used the Crick generating equations5 to produce millions of four-helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen-bond networks, and used Rosetta to connect pairs of helices with short loops and to optimize the remainder of the sequence. Of 97 such designs expressed in Escherichia coli, 65 formed constitutive heterodimers, and the crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen-bond networks. In cells, six heterodimers were fully orthogonal, and in vitro-following mixing of 32 chains from 16 heterodimer designs, denaturation in 5 M guanidine hydrochloride and reannealing-almost all of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein-based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities.
Asunto(s)
Simulación por Computador , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas/química , Proteínas/metabolismo , ADN/química , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Guanidina/farmacología , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica , Desnaturalización Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Proteínas/genéticaRESUMEN
Online citizen science projects such as GalaxyZoo1, Eyewire2 and Phylo3 have proven very successful for data collection, annotation and processing, but for the most part have harnessed human pattern-recognition skills rather than human creativity. An exception is the game EteRNA4, in which game players learn to build new RNA structures by exploring the discrete two-dimensional space of Watson-Crick base pairing possibilities. Building new proteins, however, is a more challenging task to present in a game, as both the representation and evaluation of a protein structure are intrinsically three-dimensional. We posed the challenge of de novo protein design in the online protein-folding game Foldit5. Players were presented with a fully extended peptide chain and challenged to craft a folded protein structure and an amino acid sequence encoding that structure. After many iterations of player design, analysis of the top-scoring solutions and subsequent game improvement, Foldit players can now-starting from an extended polypeptide chain-generate a diversity of protein structures and sequences that encode them in silico. One hundred forty-six Foldit player designs with sequences unrelated to naturally occurring proteins were encoded in synthetic genes; 56 were found to be expressed and soluble in Escherichia coli, and to adopt stable monomeric folded structures in solution. The diversity of these structures is unprecedented in de novo protein design, representing 20 different folds-including a new fold not observed in natural proteins. High-resolution structures were determined for four of the designs, and are nearly identical to the player models. This work makes explicit the considerable implicit knowledge that contributes to success in de novo protein design, and shows that citizen scientists can discover creative new solutions to outstanding scientific challenges such as the protein design problem.
Asunto(s)
Ciencia Ciudadana/métodos , Creatividad , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Automatización , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Juegos Experimentales , Modelos Moleculares , Interfaz Usuario-ComputadorRESUMEN
This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.
Asunto(s)
Microscopía por Crioelectrón/métodos , Modelos Moleculares , Cristalografía por Rayos X , Conformación Proteica , Proteínas/químicaRESUMEN
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Caspasa 8/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteína de Dominio de Muerte Asociada a Fas/química , Proteínas Virales/química , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Sitios de Unión , Proteínas Adaptadoras de Señalización CARD , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Microscopía por Crioelectrón , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Dominio Efector de Muerte , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Expresión Génica , Humanos , Células Jurkat , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Proteínas Virales/genética , Proteínas Virales/metabolismo , Receptor fas/farmacologíaRESUMEN
Programmed cell death protein-1 (PD-1) expressed on activated T cells inhibits T cell function and proliferation to prevent an excessive immune response, and disease can result if this delicate balance is shifted in either direction. Tumor cells often take advantage of this pathway by overexpressing the PD-1 ligand PD-L1 to evade destruction by the immune system. Alternatively, if there is a decrease in function of the PD-1 pathway, unchecked activation of the immune system and autoimmunity can result. Using a combination of computation and experiment, we designed a hyperstable 40-residue miniprotein, PD-MP1, that specifically binds murine and human PD-1 at the PD-L1 interface with a Kd of â¼100 nM. The apo crystal structure shows that the binder folds as designed with a backbone RMSD of 1.3 Å to the design model. Trimerization of PD-MP1 resulted in a PD-1 agonist that strongly inhibits murine T cell activation. This small, hyperstable PD-1 binding protein was computationally designed with an all-beta interface, and the trimeric agonist could contribute to treatments for autoimmune and inflammatory diseases.
Asunto(s)
Antígeno B7-H1/química , Receptor de Muerte Celular Programada 1/agonistas , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Antígeno B7-H1/síntesis química , Antígeno B7-H1/inmunología , Antígeno B7-H1/farmacología , Biología Computacional , Diseño de Fármacos , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/química , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/química , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.
Asunto(s)
Sustancias Macromoleculares/química , Modelos Moleculares , Proteínas/química , Programas Informáticos , Simulación del Acoplamiento Molecular , Peptidomiméticos/química , Conformación ProteicaRESUMEN
Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome-a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain. Hrd1 is the crucial membrane component for retro-translocation, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction-from the cytosol into or across membranes. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.