RESUMO
Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3' end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.
Assuntos
RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estudo de Associação Genômica Ampla , Transcrição Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Processamento de Terminações 3' de RNA/genéticaRESUMO
The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.
Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Escherichia coli , Código Genético , RNA de Transferência , Acilação , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Código Genético/genética , Hidroxiácidos/química , Hidroxiácidos/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Especificidade por Substrato , Ribossomos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
Assuntos
Regeneração Nervosa , Humanos , Neoplasias/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Isoformas de Proteínas/agonistas , Transdução de Sinais/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/química , Classe I de Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Cardiotônicos/farmacologia , Animais , Biocatálise/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Compressão Nervosa , Proliferação de Células/efeitos dos fármacosRESUMO
During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD ) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.
Assuntos
Proteínas de Ciclo Celular , Cinetocoros , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Fosforilação , Transdução de Sinais , Fuso Acromático/metabolismoRESUMO
Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.
Assuntos
Descoberta de Drogas/métodos , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Piridinas/metabolismo , Pirimidinas/metabolismo , Schistosoma mansoni/enzimologia , Tiazóis/metabolismo , Animais , Domínio Catalítico , Cristalização , Cristalografia por Raios X/métodos , Dimetil Sulfóxido/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Purina-Núcleosídeo Fosforilase/genética , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/parasitologiaRESUMO
Kinetochores are large multi-subunit complexes that attach centromeric chromatin to microtubules of the mitotic spindle, enabling sister chromatid segregation in mitosis. The inner kinetochore constitutive centromere associated network (CCAN) complex assembles onto the centromere-specific Cenp-A nucleosome (Cenp-ANuc), thereby coupling the centromere to the microtubule-binding outer kinetochore. CCAN is a conserved 14-16 subunit complex composed of discrete modules. Here, we determined the crystal structure of the Saccharomyces cerevisiae Cenp-HIKHead-TW sub-module, revealing how Cenp-HIK and Cenp-TW interact at the conserved Cenp-HIKHead-Cenp-TW interface. A major interface is formed by the C-terminal anti-parallel α-helices of the histone fold extension (HFE) of the Cenp-T histone fold domain (HFD) combining with α-helix H3 of Cenp-K to create a compact three α-helical bundle. We fitted the Cenp-HIKHead-TW sub-module to the previously determined cryo-EM map of the S. cerevisiae CCAN-Cenp-ANuc complex. This showed that the HEAT repeat domain of Cenp-IHead and C-terminal HFD of Cenp-T of the Cenp-HIKHead-TW sub-module interact with the nucleosome DNA gyre at a site close to the Cenp-ANuc dyad axis. Our structure provides a framework for understanding how Cenp-T links centromeric Cenp-ANuc to the outer kinetochore through its HFD and N-terminal Ndc80-binding motif, respectively.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Cinetocoros , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos , Proteínas de Ligação a DNA/química , Cinetocoros/química , Nucleossomos , Ligação Proteica , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Fuso AcromáticoRESUMO
The AvrRpt2 protein of the phytopathogenic bacterium Erwinia amylovora (AvrRpt2EA) is a secreted type III effector protein, which is recognised by the FB_MR5 resistance protein of Malusâ¯×â¯robusta 5, the only identified resistance protein from a Malus species preventing E. amylovora infection. The crystal structure of the immature catalytic domain of AvrRpt2EA, a C70 family cysteine protease and type III effector, was determined to a resolution of 1.85â¯Å. The structure provides insights into the cyclophilin-dependent activation of AvrRpt2, and identifies a cryptic leucine of a non-canonical cyclophilin binding motif. The structure also suggests that residue Cys156, responsible for the gene induced resistance, is not involved in substrate determination, and hints that recognition by FB_MR5 is due to direct interaction.
Assuntos
Proteínas de Bactérias/metabolismo , Erwinia amylovora/metabolismo , Malus/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cristalografia por Raios X , Erwinia amylovora/enzimologia , Interações Hospedeiro-Patógeno , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Elétrons , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Peso Molecular , Conformação Proteica , Fatores de TempoRESUMO
Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84â¯Å resolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Erwinia amylovora/enzimologia , Erwinia amylovora/metabolismo , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/metabolismo , Proteínas de Bactérias/genética , Erwinia amylovora/genética , Hexosefosfatos/metabolismo , Cinética , Rosaceae/microbiologia , Desidrogenase do Álcool de Açúcar/genética , Tomografia Computadorizada por Raios XRESUMO
The Gram-negative bacterium Erwinia amylovora is the etiological agent of fire blight, a devastating disease which affects Rosaceae such as apple, pear and quince. The siderophore desferrioxamine E plays an important role in bacterial pathogenesis by scavenging iron from the host. DfoJ, DfoA and DfoC are the enzymes responsible for desferrioxamine production starting from lysine. We have determined the crystal structures of each enzyme in the desferrioxamine E pathway and demonstrate that the biosynthesis involves the concerted action of DfoJ, followed by DfoA and lastly DfoC. These data provide the first crystal structures of a Group II pyridoxal-dependent lysine decarboxylase, a cadaverine monooxygenase and a desferrioxamine synthetase. DfoJ is a homodimer made up of three domains. Each monomer contributes to the completion of the active site, which is positioned at the dimer interface. DfoA is the first structure of a cadaverine monooxygenase. It forms homotetramers whose subunits are built by two domains: one for FAD and one for NADP+ binding, the latter of which is formed by two subdomains. We propose a model for substrate binding and the role of residues 43-47 as gate keepers for FAD binding and the role of Arg97 in cofactors turnover. DfoC is the first structure of a desferrioxamine synthetase and the first of a multi-enzyme siderophore synthetase coupling an acyltransferase domain with a Non-Ribosomal Peptide Synthetase (NRPS)-Independent Siderophore domain (NIS).
Assuntos
Erwinia amylovora/química , Ácidos Hidroxâmicos/química , Lactamas/química , Doenças das Plantas/microbiologia , Rosaceae/microbiologia , Erwinia amylovora/patogenicidade , Frutas/parasitologia , Ácidos Hidroxâmicos/metabolismo , Ferro/química , Lactamas/metabolismoRESUMO
The ability to precisely visualize the atomic geometry of the interactions between a drug and its protein target in structural models is critical in predicting the correct modifications in previously identified inhibitors to create more effective next generation drugs. It is currently common practice among medicinal chemists while attempting the above to access the information contained in three-dimensional structures by using two-dimensional projections, which can preclude disclosure of useful features. A more accessible and intuitive visualization of the three-dimensional configuration of the atomic geometry in the models can be achieved through the implementation of immersive virtual reality (VR). While bespoke commercial VR suites are available, in this work, we present a freely available software pipeline for visualising protein structures through VR. New consumer hardware, such as the HTC VIVE and the OCULUS RIFT utilized in this study, are available at reasonable prices. As an instructive example, we have combined VR visualization with fast algorithms for simulating intramolecular motions of protein flexibility, in an effort to further improve structure-led drug design by exposing molecular interactions that might be hidden in the less informative static models. This is a paradigmatic test case scenario for many similar applications in computer-aided molecular studies and design.
Assuntos
Simulação por Computador , Proteínas/química , Realidade Virtual , Algoritmos , Desenho de Fármacos , Humanos , Conformação Proteica , Software , Interface Usuário-ComputadorRESUMO
Bis-(3',5') cyclic di-guanylate (c-di-GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c-di-GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD-GYP domains. Here, we have determined the structure of an enzymatically active HD-GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c-di-GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c-di-GMP. This structure completes the picture of all domains involved in c-di-GMP metabolism and reveals that the HD-GYP family splits into two distinct subgroups containing bi- and trinuclear metal centres.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/química , Proteínas de Bactérias/química , Domínio Catalítico , GMP Cíclico/análogos & derivados , Bactérias Gram-Negativas/enzimologia , Ferro/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , GMP Cíclico/metabolismo , Evolução Molecular , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.
Assuntos
Microscopia Crioeletrônica , Cinetocoros , Proteínas Associadas aos Microtúbulos , Modelos Moleculares , Proteínas Nucleares , Humanos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Fosforilação , Aurora Quinase B/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Conformação Proteica , Proteínas Cromossômicas não HistonaRESUMO
Microtubule (MT) filaments, composed of α/ß-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/ß-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/ß-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.
Assuntos
Microscopia Crioeletrônica , Microtúbulos , Modelos Moleculares , Saccharomyces cerevisiae , Fuso Acromático , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Microtúbulos/química , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Tomografia com Microscopia Eletrônica , Conformação Proteica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/ultraestruturaRESUMO
Cell division in all domains of life requires the orchestration of many proteins, but in Archaea most of the machinery remains poorly characterized. Here we investigate the FtsZ-based cell division mechanism in Haloferax volcanii and find proteins containing photosynthetic reaction centre (PRC) barrel domains that play an essential role in archaeal cell division. We rename these proteins cell division protein B 1 (CdpB1) and CdpB2. Depletions and deletions in their respective genes cause severe cell division defects, generating drastically enlarged cells. Fluorescence microscopy of tagged FtsZ1, FtsZ2 and SepF in CdpB1 and CdpB2 mutant strains revealed an unusually disordered divisome that is not organized into a distinct ring-like structure. Biochemical analysis shows that SepF forms a tripartite complex with CdpB1/2 and crystal structures suggest that these two proteins might form filaments, possibly aligning SepF and the FtsZ2 ring during cell division. Overall our results indicate that PRC-domain proteins play essential roles in FtsZ-based cell division in Archaea.
Assuntos
Haloferax volcanii , Complexo de Proteínas do Centro de Reação Fotossintética , Divisão Celular , Citoesqueleto , Haloferax volcanii/genética , Microscopia de FluorescênciaRESUMO
Bacteriophytochromes (BphPs) are biliverdin IXα-containing photoreceptors that photoconvert between red (Pr) and far-red (Pfr) absorbing states. BphPs are one half of a two-component system that transmits a light signal to a histidine kinase domain and then to a gene-response regulator. In Rhodopseudomonas palustris, synthesis of a light-harvesting complex (LH4) is controlled by two BphPs (RpBphP2 and RpBphP3). Despite their high sequence identity (52%), their absorption spectra are very different. The spectra of RpBphP2 exhibit classic Pr-to-Pfr photoconversion, whereas RpBphP3 quenches and a high-energy Pnr state emerges [Giraud et al. (2005), J. Biol. Chem. 280, 32389-32397]. Crystallization of the chromophore-binding domain (CBD) of RpBphP2 (RpBphP2-CBD) proved to be difficult and the structure of RpBphP3-CBD was used to crystallize RpBphP2-CBD* using homologue-directed mutagenesis. The structure shows that dimerization is an important factor in successful crystallization of RpBphP2-CBD* and arises from an N136R mutation. Mutations at this site correlate with an ability to dimerize in other truncated BphPs and may also be important for full-length dimer formation. Comparison of the RpBphP3-CBD and RpBphP2-CBD* biliverdin IXα pockets revealed that the former has additional hydrogen bonding around the B and D pyrrole rings that may constrain photoconversion to Pfr, resulting in a strained photoexcited Pnr state.
Assuntos
Proteínas de Bactérias/química , Cromatóforos/química , Complexos de Proteínas Captadores de Luz/química , Fitocromo/química , Cromatografia em Gel , Clonagem Molecular , Cristalização , Cristalografia por Raios X/métodos , Dimerização , Modelos Químicos , Conformação Molecular , Mutagênese , Mutação , Estrutura Terciária de Proteína , Rodopseudomonas/metabolismo , Espectrofotometria Ultravioleta/métodosRESUMO
In X-ray macromolecular crystallography, cryoprotection of crystals mounted on harvesting loops is achieved when the water in the sample solvent transitions to vitreous ice before crystalline ice forms. This is achieved by rapid cooling in liquid nitro-gen or propane. Protocols for protein crystal cryoprotection are based on either increasing the environmental pressure or reducing the water fraction in the solvent. This study presents a new protocol for cryoprotecting crystals. It is based on vapour diffusion dehydration of the crystal drop to reduce the water fraction in the solvent by adding a highly concentrated salt solution, 13â M potassium formate (KF13), directly to the reservoir. Several salt solutions were screened to identify KF13 as optimal. Cryoprotection using the KF13 protocol is non-invasive to the crystal, high throughput and easy to implement, can benefit diffraction resolution and ligand binding, and is very useful in cases with high redundancy such as drug-discovery projects which use very large compound or fragment libraries. An application of KF13 to discover new crystal hits from clear drops of equilibrated crystallization screening plates is also shown.
RESUMO
The success and speed of atomic structure determination of biological macromolecules by X-ray crystallography depends critically on the availability of diffraction-quality crystals. However, the process of screening crystallization conditions often consumes large amounts of sample and time. An innovative protein crystallization screen formulation called FUSION has been developed to help with the production of useful crystals. The concept behind the formulation of FUSION was to combine the most efficient components from the three MORPHEUS screens into a single screen using a systematic approach. The resulting formulation integrates 96 unique combinations of crystallization additives. Most of these additives are small molecules and ions frequently found in crystal structures of the Protein Data Bank (PDB), where they bind proteins and complexes. The efficiency of FUSION is demonstrated by obtaining high yields of diffraction-quality crystals for seven different test proteins. In the process, two crystal forms not currently in the PDB for the proteins α-amylase and avidin were discovered.
RESUMO
Kinetochores assemble onto specialized centromeric CENP-A (centromere protein A) nucleosomes (CENP-ANuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-electron microscopy structures of the human inner kinetochore constitutive centromere associated network (CCAN) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, and a linker DNA segment of the α-satellite repeat emerges from the fully wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.
Assuntos
Proteína Centromérica A , Cinetocoros , Nucleossomos , Centrômero/química , Proteína Centromérica A/química , Microscopia Crioeletrônica , DNA/química , Humanos , Cinetocoros/química , Nucleossomos/química , Ligação ProteicaRESUMO
The ESCRT machinery, comprising of multiple proteins and subcomplexes, is crucial for membrane remodelling in eukaryotic cells, in processes that include ubiquitin-mediated multivesicular body formation, membrane repair, cytokinetic abscission, and virus exit from host cells. This ESCRT system appears to have simpler, ancient origins, since many archaeal species possess homologues of ESCRT-III and Vps4, the components that execute the final membrane scission reaction, where they have been shown to play roles in cytokinesis, extracellular vesicle formation and viral egress. Remarkably, metagenome assemblies of Asgard archaea, the closest known living relatives of eukaryotes, were recently shown to encode homologues of the entire cascade involved in ubiquitin-mediated membrane remodelling, including ubiquitin itself, components of the ESCRT-I and ESCRT-II subcomplexes, and ESCRT-III and Vps4. Here, we explore the phylogeny, structure, and biochemistry of Asgard homologues of the ESCRT machinery and the associated ubiquitylation system. We provide evidence for the ESCRT-I and ESCRT-II subcomplexes being involved in ubiquitin-directed recruitment of ESCRT-III, as it is in eukaryotes. Taken together, our analyses suggest a pre-eukaryotic origin for the ubiquitin-coupled ESCRT system and a likely path of ESCRT evolution via a series of gene duplication and diversification events.