RESUMO
Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.
Assuntos
Anticorpos Neutralizantes/imunologia , HIV-1/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Polissacarídeos/imunologia , SARS-CoV-2/imunologia , Vírus da Imunodeficiência Símia/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Dimerização , Epitopos/imunologia , Glicosilação , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Macaca mulatta , Polissacarídeos/química , Receptores de Antígenos de Linfócitos B/química , Vírus da Imunodeficiência Símia/genética , Vacinas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/imunologia , Bacteriófagos/genética , Bacteriófagos/imunologia , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/ultraestrutura , DNA Viral/química , Modelos Químicos , Modelos Moleculares , Complexos Multiproteicos/química , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/ultraestruturaRESUMO
Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ â¼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.
Assuntos
Descoberta de Drogas , Glutamato Desidrogenase/ultraestrutura , Isocitrato Desidrogenase/ultraestrutura , L-Lactato Desidrogenase/ultraestrutura , Aminoquinolinas/química , Aminoquinolinas/farmacologia , Animais , Bovinos , Galinhas , Microscopia Crioeletrônica , Cristalografia por Raios X , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/química , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/química , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/química , Modelos Moleculares , Conformação Proteica , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD â¼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (â¼ 10-25 Å) by hinge-like motions as large as â¼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.
Assuntos
Proteínas de Bactérias/ultraestrutura , Proteínas de Transporte de Cátions/ultraestrutura , Magnésio/metabolismo , Thermotoga maritima/química , Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Microscopia Crioeletrônica , Modelos Moleculares , Simulação de Dinâmica MolecularRESUMO
The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. MglA-SspA assembles with the σ70-associated RNAP holoenzyme (RNAPσ70), forming a virulence-specialized polymerase. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence, but the mechanism is unknown. Here we report FtRNAPσ70-promoter-DNA, FtRNAPσ70-(MglA-SspA)-promoter DNA, and FtRNAPσ70-(MglA-SspA)-ppGpp-PigR-promoter DNA cryo-EM structures. Structural and genetic analyses show MglA-SspA facilitates σ70 binding to DNA to regulate virulence and virulence-enhancing genes. Our Escherichia coli RNAPσ70-homodimeric EcSspA structure suggests this is a general SspA-transcription regulation mechanism. Strikingly, our FtRNAPσ70-(MglA-SspA)-ppGpp-PigR-DNA structure reveals ppGpp binding to MglA-SspA tethers PigR to promoters. PigR in turn recruits FtRNAP αCTDs to DNA UP elements. Thus, these studies unveil a unique mechanism for Ft pathogenesis involving a virulence-specialized RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.
Assuntos
RNA Polimerases Dirigidas por DNA , Francisella tularensis , Regiões Promotoras Genéticas , Fator sigma , Fatores de Virulência , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Francisella tularensis/genética , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidade , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid1-4. Despite the important role of NPR1 in plant immunity5-7, understanding of its regulatory mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding domain. Crystal structure analysis reveals a unique zinc-finger motif in BTB for interacting with ankyrin repeats and mediating NPR1 oligomerization. We found that, after stimulation, salicylic-acid-induced folding and docking of the salicylic-acid-binding domain onto ankyrin repeats is required for the transcriptional cofactor activity of NPR1, providing a structural explanation for a direct role of salicylic acid in regulating NPR1-dependent gene expression. Moreover, our structure of the TGA32-NPR12-TGA32 complex, DNA-binding assay and genetic data show that dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form an enhanceosome. The stepwise assembly of the NPR1-TGA complex suggests possible hetero-oligomeric complex formation with other transcription factors, revealing how NPR1 reprograms the defence transcriptome.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Microscopia Crioeletrônica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Ácido Salicílico/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement toward the catalytic pocket by the N terminus upon ligand binding and identifies residues required for substrate binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved among Cryptococcal species and closely related basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.
Assuntos
Antifúngicos , Cryptococcus neoformans , Glucosiltransferases , Trealose , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/genética , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Antifúngicos/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Trealose/metabolismo , Trealose/análogos & derivados , Trealose/biossíntese , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Modelos Moleculares , Humanos , Domínio Catalítico , Cristalografia por Raios XRESUMO
Single-particle cryo-electron tomography is an emerging technique capable of determining the structure of proteins imaged within the native context of cells at molecular resolution. While high-throughput techniques for sample preparation and tilt-series acquisition are beginning to provide sufficient data to allow structural studies of proteins at physiological concentrations, the complex data analysis pipeline and the demanding storage and computational requirements pose major barriers for the development and broader adoption of this technology. Here, we present a scalable, end-to-end framework for single-particle cryo-electron tomography data analysis from on-the-fly pre-processing of tilt series to high-resolution refinement and classification, which allows efficient analysis and visualization of datasets with hundreds of tilt series and hundreds of thousands of particles. We validate our approach using in vitro and cellular datasets, demonstrating its effectiveness at achieving high-resolution and revealing conformational heterogeneity in situ. The framework is made available through an intuitive and easy-to-use computer application, nextPYP ( http://nextpyp.app ).
Assuntos
Tomografia com Microscopia Eletrônica , Software , Tomografia com Microscopia Eletrônica/métodos , Microscopia Crioeletrônica/métodos , Proteínas , Processamento de Imagem Assistida por Computador/métodosRESUMO
In the PDF version of this Article, owing to a typesetting error, an incorrect figure was used for Extended Data Fig. 5; the correct figure was used in the HTML version. This has been corrected online.
RESUMO
G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, Gi-bound and arrestin-bound forms of rhodopsin with inactive and Gs-bound forms of the ß2-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of Gs, Gi and arrestin by activated G-protein-coupled receptors.
Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Rodopsina/metabolismo , Rodopsina/ultraestrutura , Arrestina/química , Arrestina/metabolismo , Sítios de Ligação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Modelos Moleculares , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Rodopsina/química , Transdução de Sinais , Especificidade por SubstratoRESUMO
Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.
Assuntos
Estresse Oxidativo , Ribossomos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , MutaçãoRESUMO
Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.
Assuntos
Microscopia Crioeletrônica , Receptores de AMPA/metabolismo , Receptores de AMPA/ultraestrutura , Receptores de Ácido Caínico/metabolismo , Receptores de Ácido Caínico/ultraestrutura , Animais , Sítios de Ligação , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Modelos Moleculares , Estrutura Terciária de Proteína/efeitos dos fármacos , Ratos , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/química , Receptores de Ácido Caínico/química , Receptor de GluK2 CainatoRESUMO
In recent years, an abundance of new molecular structures have been elucidated using cryo-electron microscopy (cryo-EM), largely due to advances in hardware technology and data processing techniques. Owing to these new exciting developments, cryo-EM was selected by Nature Methods as Method of the Year 2015, and the Nobel Prize in Chemistry 2017 was awarded to three pioneers in the field. The main goal of this article is to introduce the challenging and exciting computational tasks involved in reconstructing 3-D molecular structures by cryo-EM. Determining molecular structures requires a wide range of computational tools in a variety of fields, including signal processing, estimation and detection theory, high-dimensional statistics, convex and non-convex optimization, spectral algorithms, dimensionality reduction, and machine learning. The tools from these fields must be adapted to work under exceptionally challenging conditions, including extreme noise levels, the presence of missing data, and massively large datasets as large as several Terabytes. In addition, we present two statistical models: multi-reference alignment and multi-target detection, that abstract away much of the intricacies of cryo-EM, while retaining some of its essential features. Based on these abstractions, we discuss some recent intriguing results in the mathematical theory of cryo-EM, and delineate relations with group theory, invariant theory, and information theory.
RESUMO
We report the solution structure of Escherichia coli ß-galactosidase (â¼465 kDa), solved at â¼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg(2+) ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of â¼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e(-)/Å(2). In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , beta-Galactosidase/química , beta-Galactosidase/ultraestrutura , Fenômenos Biofísicos , Domínio Catalítico , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/enzimologia , Modelos Moleculares , Conformação Proteica , Estrutura Quaternária de Proteína , Eletricidade EstáticaRESUMO
Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.
Assuntos
Microscopia Crioeletrônica/métodos , Glutamato Desidrogenase/ultraestrutura , Animais , Bovinos , Cristalografia por Raios X , Glutamato Desidrogenase/química , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Ligantes , Mamíferos , Modelos Moleculares , NAD/química , NAD/metabolismo , Estrutura Quaternária de ProteínaRESUMO
The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP.
Assuntos
Biocatálise , Microscopia Crioeletrônica , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/ultraestrutura , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Cristalografia por Raios X , Humanos , Hidrólise , Modelos Biológicos , Conformação ProteicaRESUMO
Glutamate receptor ion channels are membrane proteins that mediate excitatory synaptic transmission in the central nervous system of vertebrates. Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states. Here, we report the use of single-particle cryoelectron tomography to determine the structures, at â¼21 Å resolution, of full-length GluK2 kainate receptors trapped in antagonist-bound resting and agonist-bound desensitized states. The resting state, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2, with well-resolved proximal and distal subunits exhibiting cross-over between the twofold symmetric amino terminal domain and a twofold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD undergoes a major rearrangement, resulting in a separation of the four subunits by â¼25 Å. However, the amino terminal domain, transmembrane, and cytoplasmic regions of the receptor have similar conformations in the resting and desensitized states. The LBD rearrangement was not anticipated in prior models based on crystal structures for soluble LBD dimer assemblies, and we speculate that subunit separation allows a better match to the fourfold symmetric ion channel domain. From fits of the amino terminal domain and LBD domains into the density map of the desensitized state we have derived a structural model for differences in quaternary conformation between the resting and desensitized states.
Assuntos
Modelos Moleculares , Receptores de Ácido Caínico/química , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Ativação do Canal Iônico , Ligantes , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Ratos , Tomografia , Receptor de GluK2 CainatoRESUMO
The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function.
Assuntos
Ebolavirus/metabolismo , Doença pelo Vírus Ebola/virologia , Proteínas do Envelope Viral/química , Microscopia Crioeletrônica , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/ultraestrutura , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
We describe cryo-electron microscopic studies of the interaction between the ectodomain of the trimeric HIV-1 envelope glycoprotein (Env) and Z13e1, a broadly neutralizing antibody that targets the membrane-proximal external region (MPER) of the gp41 subunit. We show that Z13e1-bound Env displays an open quaternary conformation similar to the CD4-bound conformation. Our results support the idea that MPER-directed antibodies, such as Z13e1, block viral entry by interacting with Env at a step after CD4 activation.