RESUMO
Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCA's binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.
Assuntos
Anidrases Carbônicas , Ribulose-Bifosfato Carboxilase , Ribulose-Bifosfato Carboxilase/metabolismo , Anidrases Carbônicas/metabolismo , Dióxido de Carbono/metabolismo , Microscopia Crioeletrônica , Organelas/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Quality control mechanisms intervene appropriately when defective translation events occur, in order to preserve the integrity of protein synthesis. Rescue of ribosomes translating on messenger RNAs that lack stop codons is one of the co-translational quality control pathways. In many bacteria, ArfA recognizes stalled ribosomes and recruits the release factor RF2, which catalyses the termination of protein synthesis. Although an induced-fit mechanism of nonstop mRNA surveillance mediated by ArfA and RF2 has been reported, the molecular interaction between ArfA and RF2 in the ribosome that is responsible for the mechanism is unknown. Here we report an electron cryo-microscopy structure of ArfA and RF2 in complex with the 70S ribosome bound to a nonstop mRNA. The structure, which is consistent with our kinetic and biochemical data, reveals the molecular interactions that enable ArfA to specifically recruit RF2, not RF1, into the ribosome and to enable RF2 to release the truncated protein product in this co-translational quality control pathway. The positively charged C-terminal domain of ArfA anchors in the mRNA entry channel of the ribosome. Furthermore, binding of ArfA and RF2 induces conformational changes in the ribosomal decoding centre that are similar to those seen in other protein-involved decoding processes. Specific interactions between residues in the N-terminal domain of ArfA and RF2 help RF2 to adopt a catalytically competent conformation for peptide release. Our findings provide a framework for understanding recognition of the translational state of the ribosome by new proteins, and expand our knowledge of the decoding potential of the ribosome.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Biocatálise , Códon de Terminação , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Modelos Moleculares , Fatores de Terminação de Peptídeos/ultraestrutura , Ligação Proteica , Domínios Proteicos , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Ribossomos/química , Ribossomos/ultraestruturaRESUMO
The human CDK-activating kinase (CAK), composed of CDK7, cyclin H, and MAT1, is involved in the control of transcription initiation and the cell cycle. Because of these activities, it has been identified as a promising target for cancer chemotherapy. A number of CDK7 inhibitors have entered clinical trials, among them ICEC0942 (also known as CT7001). Structural information can aid in improving the affinity and specificity of such drugs or drug candidates, reducing side effects in patients. Here, we have determined the structure of the human CAK in complex with ICEC0942 at 2.5 Å-resolution using cryogenic electron microscopy. Our structure reveals conformational differences of ICEC0942 compared with previous X-ray crystal structures of the CDK2-bound complex, and highlights the critical ability of cryogenic electron microscopy to resolve structures of drug-bound protein complexes without the need to crystalize the protein target.
Assuntos
Quinases Ciclina-Dependentes , Ciclo Celular , Divisão Celular , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Humanos , Fosforilação , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
A strategy that utilizes DNA for controlling the association pathway of proteins is described. This strategy uses sequence-specific DNA interactions to program energy barriers for polymerization, allowing for either step-growth or chain-growth pathways to be accessed. Two sets of mutant green fluorescent protein (mGFP)-DNA monomers with single DNA modifications have been synthesized and characterized. Depending on the deliberately controlled sequence and conformation of the appended DNA, these monomers can be polymerized through either a step-growth or chain-growth pathway. Cryo-electron microscopy with Volta phase plate technology enables the visualization of the distribution of the oligomer and polymer products, and even the small mGFP-DNA monomers. Whereas cyclic and linear polymer distributions were observed for the step-growth DNA design, in the case of the chain-growth system linear chains exclusively were observed, and a dependence of the chain length on the concentration of the initiator strand was noted. Importantly, the chain-growth system possesses a living character whereby chains can be extended with the addition of fresh monomer. This work represents an important and early example of mechanistic control over protein assembly, thereby establishing a robust methodology for synthesizing oligomeric and polymeric protein-based materials with exceptional control over architecture.
Assuntos
DNA/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Mutação , Tamanho da Partícula , PolimerizaçãoRESUMO
ß-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease.
Assuntos
Sítio Alostérico , Glucosilceramidase/química , Glucosilceramidase/metabolismo , Multimerização Proteica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Cristalografia por Raios X , Fibroblastos/metabolismo , Glucosilceramidase/genética , Células HEK293 , Humanos , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , MutaçãoRESUMO
The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 µm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Biofilmes , Matriz Extracelular/ultraestrutura , Myxococcus xanthus/química , Membrana Celular/ultraestrutura , Microscopia Eletrônica de Varredura , Myxococcus xanthus/fisiologia , Myxococcus xanthus/ultraestruturaRESUMO
Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 Å resolution or higher. When we imaged apoF with a laser phase plate (LPP), however, we observed more severe particle-to-particle variation in the images than we had previously thought to exist. Similarly, we found that images of ribulose bisphosphate carboxylase/oxygenase (rubisco) also exhibited a much greater amount of heterogeneity than expected. By comparison to simulations of images, we verified that the heterogeneity is not explained by the known features of the LPP, shot noise, or differences in particle orientation. We also demonstrate that our specimens are comparable to those previously used in the literature, based on using the final-reconstruction resolution as the metric for evaluation. All of this leads us to the hypothesis that the heterogeneity is due to damage that has occurred either during purification of the specimen or during preparation of the grids. It is not, however, our goal to explain the causes of heterogeneity; rather, we report that using the LPP has made the apparent damage too obvious to be ignored. In hindsight, similar heterogeneity can be seen in images of apoF and the 20S proteasome which others had recorded with a Volta phase plate. We therefore conclude that the increased contrast of phase-plate images (at low spatial frequencies) should also make it possible to visualize, on a single-particle basis, various forms of biologically functional heterogeneity in structure that had previously gone unnoticed. HIGHLIGHTS: Phase plates recover low-frequency information with significantly improved SNRLaser phase-plate images reveal unexpected amounts of structural heterogeneityIn retrospect, similar heterogeneity can also be seen in Volta phase-plate imagesParticle heterogeneity produces "structural noise", which may diminish map quality.
RESUMO
Recognizing that interaction with the air-water interface (AWI) is a major challenge for cryo-EM, we first review current approaches designed to avoid it. Of these, immobilizing particles on affinity grids is arguably the most promising. In addition, we review efforts to gain more reliable control of the sample thicknesses, not the least important reason being to prevent immobilized particles from coming in contact with the AWI of the remaining buffer. It is emphasized that avoiding such a contact is as important for cryo-ET as for single-particle cryo-EM. Finally, looking to the future, it is proposed that immobilized samples might be used to perform time-resolved biochemical experiments directly on EM grids rather than just in test tubes or cuvettes.
Assuntos
Água , Microscopia CrioeletrônicaRESUMO
We identify thermal magnetic field fluctuations, caused by thermal electron motion ("Johnson noise") in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.
RESUMO
We identify thermal magnetic field fluctuations, caused by thermal electron motion ("Johnson noise") in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.
RESUMO
Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1-GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1-GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Análise por Conglomerados , Modelos Animais de Doenças , Proteína Relacionada a TNFR Induzida por Glucocorticoide/agonistas , Humanos , Imunoterapia/métodos , Camundongos , Neoplasias/tratamento farmacológico , Receptores do Fator de Necrose Tumoral/agonistas , Linfócitos TRESUMO
Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. C9orf72 forms a complex with SMCR8 and WDR41, which was reported to have GTPase activating protein activity toward ARF proteins, RAB8A, and RAB11A. We determined the cryo-EM structure of ARF1-GDP-BeF3- bound to C9orf72:SMCR8:WDR41. The SMCR8longin and C9orf72longin domains form the binding pocket for ARF1. One face of the C9orf72longin domain holds ARF1 in place, while the SMCR8longin positions the catalytic finger Arg147 in the ARF1 active site. Mutations in interfacial residues of ARF1 and C9orf72 reduced or eliminated GAP activity. RAB8A GAP required ~10-fold higher concentrations of the C9orf72 complex than for ARF1. These data support a specific function for the C9orf72 complex as an ARF GAP. The structure also provides a model for the active forms of the longin domain GAPs of FLCN and NPRL2 that regulate the Rag GTPases of the mTORC1 pathway.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína C9orf72/metabolismo , Proteínas de Transporte/metabolismo , Demência Frontotemporal/genética , Proteínas rab de Ligação ao GTP/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteína C9orf72/genética , Proteínas de Transporte/genética , Microscopia Crioeletrônica , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/genética , Estrutura Terciária de Proteína/genéticaRESUMO
SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.
Assuntos
Microscopia Crioeletrônica , Nanoestruturas , SARS-CoV-2 , Proteínas Viroporinas/química , Proteínas Viroporinas/ultraestrutura , Animais , Cálcio/metabolismo , Quirópteros/virologia , Coronaviridae , Eletrofisiologia , Fluorescência , Humanos , Transporte de Íons , Lipossomos , Modelos Moleculares , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fases de Leitura Aberta , Imagem Óptica , Reprodutibilidade dos Testes , SARS-CoV-2/química , SARS-CoV-2/ultraestrutura , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/ultraestrutura , Proteínas Viroporinas/antagonistas & inibidoresRESUMO
Transmission electron microscopy (TEM) of vitrified biological macromolecules (cryo-EM) is limited by the weak phase contrast signal that is available from such samples. Using a phase plate would thus substantially improve the signal-to-noise ratio. We have previously demonstrated the use of a high-power Fabry-Perot cavity as a phase plate for TEM. We now report improvements to our laser cavity that allow us to achieve record continuous wave intensities of over 450 GW/cm2, sufficient to produce the optimal 90° phase shift for 300 keV electrons. In addition, we have performed the first cryo-EM reconstruction using a laser phase plate, demonstrating that the stability of this laser phase plate is sufficient for use during standard cryo-EM data collection.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a1,2. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma3-6. 3a has been implicated in viral release7, inhibition of autophagy8, inflammasome activation9, and cell death10,11 and its deletion reduces viral titer and morbidity in mice1, raising the possibility that 3a could be an effective vaccine or therapeutic target3,12. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 Å resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.
RESUMO
Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.
Assuntos
Biofilmes/crescimento & desenvolvimento , Tomografia com Microscopia Eletrônica/métodos , Imageamento Tridimensional , Myxococcus xanthus/ultraestrutura , Cromossomos Bacterianos/ultraestrutura , Vesículas Citoplasmáticas/ultraestrutura , Fímbrias Bacterianas/ultraestrutura , Myxococcus xanthus/fisiologiaRESUMO
Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.
Assuntos
Proteínas de Bactérias/metabolismo , Espectrometria de Massas/métodos , Methylococcaceae/metabolismo , Modelos Moleculares , Oxigenases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cobre/química , Cobre/metabolismo , Microscopia Crioeletrônica , Metano/metabolismo , Metanol/metabolismo , Methylococcaceae/química , Methylococcaceae/ultraestrutura , Oxirredução , Oxigenases/química , Oxigenases/ultraestrutura , Processamento de Proteína Pós-TraducionalRESUMO
The aqua (glycero) porins conduct water (and glycerol) across cell membranes. The structure of these channels reveals a tripathic channel that supports a hydrophobic surface and, opposite to this, a line of eight hydrogen-bond acceptors and four hydrogen-bond donors. The eight carbonyls act as acceptors for water (or glycerol OH) molecules. The central water molecule in the channel is oriented to polarize hydrogen atoms outward from the center. This arrangement suggests how the structure prevents the potentially lethal conduction of protons across the membrane. The structure also suggests the mechanism behind the selectivity of aquaglyceroporins for glycerol, the basis for enantioselectivity among alditols, and the basis for the prevention of any leakage of the electrochemical gradient.