RESUMO
The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialisation in development and disease. However, the inability to accurately visualise these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualise post-transcriptional modifications within the 18S rRNA and four post-translational modifications of ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilisation and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unravelling the functional role of ribosomal RNA modifications.
Assuntos
Proteínas Ribossômicas , Subunidades Ribossômicas Menores de Eucariotos , Humanos , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Microscopia Crioeletrônica , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/metabolismoRESUMO
Natural products that target the eukaryotic ribosome are promising therapeutics to treat a variety of cancers. It is therefore essential to determine their molecular mechanism of action to fully understand their mode of interaction with the target and to inform the development of new synthetic compounds with improved potency and reduced cytotoxicity. Toward this goal, we have previously established a short synthesis pathway that grants access to multiple congeners of the lissoclimide family. Here we present the X-ray co-crystal structure at 3.1 Å resolution of C45, a potent congener with two A-ring chlorine-bearing stereogenic centers with 'unnatural' configurations, with the yeast 80S ribosome, intermolecular interaction energies of the C45/ribosome complex, and single-molecule FRET data quantifying the impact of C45 on both human and yeast ribosomes. Together, these data provide new insights into the role of unusual non-covalent halogen bonding interactions involved in the binding of this synthetic compound to the 80S ribosome.
Assuntos
Produtos Biológicos/química , Diterpenos/química , Modelos Moleculares , Ribossomos/química , Succinimidas/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Diterpenos/síntese química , Células Eucarióticas/química , Humanos , Ligação Proteica , RNA Ribossômico/química , RNA Ribossômico/genética , Ribossomos/genética , Saccharomyces cerevisiae/química , Succinimidas/síntese químicaRESUMO
The ability of ribosomes to translate the genetic code into protein requires a finely tuned ion and solvent ecosystem. However, the lack of high-resolution structures has precluded accurate positioning of all the functional elements of the ribosome and limited our understanding of the specific role of ribosomal RNA chemical modifications in modulating ribosome function in health and disease. Here, using a new sample preparation methodology based on functionalised pristine graphene-coated grids, we solve the cryo-EM structure of the human large ribosomal subunit to a resolution of 1.67 Å. The accurate assignment of water molecules, magnesium and potassium ions in our model highlights the fundamental biological role of ribosomal RNA methylation in harnessing unconventional carbon-oxygen hydrogen bonds to establish chemical interactions with the environment and fine-tune the functional interplay with tRNA. In addition, the structures of three translational inhibitors bound to the human large ribosomal subunit at better than 2 Å resolution provide mechanistic insights into how three key druggable pockets of the ribosome are targeted and illustrate the potential of this methodology to accelerate high-throughput structure-based design of anti-cancer therapeutics.
RESUMO
Angiotensin-converting enzyme 2 (ACE2) is a metalloprotease that cleaves angiotensin II, a peptide substrate involved in the regulation of hypertension. Here, we identified a series of constrained bicyclic peptides, Bicycle, inhibitors of human ACE2 by panning highly diverse bacteriophage display libraries. These were used to generate X-ray crystal structures which were used to inform the design of additional Bicycles with increased affinity and inhibition of ACE2 enzymatic activity. This novel structural class of ACE2 inhibitors is among the most potent ACE2 inhibitors yet described in vitro, representing a valuable tool to further probe ACE2 function and for potential therapeutic utility.
Assuntos
Enzima de Conversão de Angiotensina 2 , Carboxipeptidases , Humanos , Carboxipeptidases/química , Peptidil Dipeptidase A , Ciclismo , Peptídeos/farmacologia , Angiotensina II , Fragmentos de PeptídeosRESUMO
Deinococcus radiodurans is well known for its extreme tolerance to harsh conditions and for its extraordinary ability to repair DNA. Double-strand breaks (DSBs) are the most hazardous lesions that can be induced by ionizing radiation, and homologous recombination (HR) is the principal mechanism by which the integrity of the DNA is restored. In D. radiodurans the RecFOR complex is the main actor in HR and the RecN protein is believed to play an important role in DSB recognition. Here, SAXS and preliminary X-ray diffraction studies are presented of the head domain, which is the globular region formed upon interaction of the N- and C-terminal domains of RecN. The crystal structure of this domain was solved using the single-wavelength anomalous dispersion method. Model building and refinement are in progress.
Assuntos
Proteínas de Bactérias/química , Enzimas de Restrição do DNA/química , Deinococcus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/isolamento & purificação , Expressão Gênica , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Deinococcus radiodurans has developed an efficient mechanism which allows the integrity of its entire genome to be fully restored after exposure to very high doses of ionizing radiation. Homologous recombination plays a crucial role in this process. RecN is a protein that belongs to the SMC-like protein family and is suggested to be involved in DNA repair. RecN is composed of a globular domain and an antiparallel coiled-coil region which connects the N- and C-termini. It has been suggested that dimerization of RecN occurs via the coiled-coil domain, but to date there is no structural or biochemical evidence for this. Here, SAXS studies and preliminary X-ray diffraction data of crystals of the purified coiled-coil domain of RecN are presented. The structure was solved by single-wavelength anomalous dispersion using SeMet derivatives, and preliminary electron-density maps support the rod-like model derived from the SAXS data. Model building and refinement are still ongoing.
Assuntos
Proteínas de Bactérias/química , Enzimas de Restrição do DNA/química , Deinococcus/enzimologia , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Enzimas de Restrição do DNA/isolamento & purificação , Expressão Gênica , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Many cellular processes, including ribosome biogenesis, are regulated through post-transcriptional RNA modifications. Here, a genome-wide analysis of the human mitochondrial transcriptome shows that 2'-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, introduced by MRM1, MRM2 and MRM3, with the modifications installed by the latter two proteins being interdependent. MRM2 controls mitochondrial respiration by regulating mitoribosome biogenesis. In its absence, mtLSU particles (visualized by cryo-EM at the resolution of 2.6 Å) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module, allowing five mtLSU biogenesis intermediates with different intersubunit interface configurations to be placed along the assembly pathway. However, mitoribosome biogenesis does not depend on the methyltransferase activity of MRM2. Disruption of the MRM2 Drosophila melanogaster orthologue leads to mitochondria-related developmental arrest. This work identifies a key checkpoint during mtLSU assembly, essential to maintain mitochondrial homeostasis.
Assuntos
Proteínas de Drosophila/metabolismo , Metiltransferases/metabolismo , Ribossomos Mitocondriais/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Masculino , Metilação , Metiltransferases/genética , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
Protein biosynthesis is a vital process for all kingdoms of life. The ribosome is the massive ribonucleoprotein machinery that reads the genetic code, in the form of messenger RNA (mRNA), to produce proteins. The mechanism of translation is tightly regulated to ensure that cell growth is well sustained. Because of the central role fulfilled by the ribosome, it is not surprising that halting its function can be detrimental and incompatible with life. In bacteria, the ribosome is a major target of inhibitors, as demonstrated by the high number of small molecules identified to bind to it. In eukaryotes, the design of ribosome inhibitors may be used as a therapy to treat cancer cells, which exhibit higher proliferation rates compared to healthy ones. Exciting experimental achievements gathered during the last few years confirmed that the ribosome indeed represents a relevant platform for the development of anticancer drugs. We provide herein an overview of the latest structural data that helped to unveil the molecular bases of inhibition of the eukaryotic ribosome triggered by small molecules.
RESUMO
Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and ex vivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration.
Assuntos
Biossíntese de Proteínas , Ribossomos , Células-Tronco Hematopoéticas , Ribossomos/metabolismoRESUMO
For the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress-induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit. Here we describe the binding mode of Staphylococcus aureus RsfS to the large ribosomal subunit and present a 3.2 Å resolution cryo-EM reconstruction of the 50S-RsfS complex together with the crystal structure of uL14-RsfS complex solved at 2.3 Å resolution. The understanding of the detailed landscape of RsfS-uL14 interactions within the ribosome shed light on the mechanism of ribosome shutdown in the human pathogen S. aureus and might deliver a novel target for pharmacological drug development and treatment of bacterial infections.
Assuntos
Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenvolvimento de Medicamentos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Subunidades RibossômicasRESUMO
During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.
Assuntos
Peptidil Transferases/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , Peptidil Transferases/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/ultraestruturaRESUMO
One of the most critical steps of protein biosynthesis is the coupled movement of mRNA, which encodes genetic information, with tRNAs on the ribosome. In eukaryotes, this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-electron microscopy structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights into the role of diphthamide in the mechanism of translation fidelity in eukaryotes.
Assuntos
Guanosina Trifosfato/metabolismo , Histidina/análogos & derivados , Fator 2 de Elongação de Peptídeos/química , Biossíntese de Proteínas , RNA Mensageiro/química , RNA de Transferência/química , Ribossomos/química , Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , Histidina/química , Histidina/metabolismo , Hidrólise , Modelos Moleculares , Fator 2 de Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismoRESUMO
Alkaloids isolated from the Amaryllidaceae plants have potential as therapeutics for treating human diseases. Haemanthamine has been studied as a novel anticancer agent due to its ability to overcome cancer cell resistance to apoptosis. Biochemical experiments have suggested that hemanthamine targets the ribosome. However, a structural characterization of its mechanism has been missing. Here we present the 3.1 Å resolution X-ray structure of haemanthamine bound to the Saccharomyces cerevisiae 80S ribosome. This structure reveals that haemanthamine targets the A-site cleft on the large ribosomal subunit rearranging rRNA to halt the elongation phase of translation. Furthermore, we provide evidence that haemanthamine and other Amaryllidaceae alkaloids also inhibit specifically ribosome biogenesis, triggering nucleolar stress response and leading to p53 stabilization in cancer cells. Together with a computer-aided interpretation of existing structure-activity relationships of Amaryllidaceae alkaloids congeners, we provide a rationale for designing molecules with enhanced potencies and reduced toxicities.
Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Colo/metabolismo , Fenantridinas/farmacologia , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Alcaloides de Amaryllidaceae/química , Antineoplásicos/química , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Cristalografia por Raios X , Células HCT116 , Humanos , Modelos Moleculares , Conformação Molecular , Fenantridinas/química , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Ribossomos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismoRESUMO
The lissoclimides are unusual succinimide-containing labdane diterpenoids that were reported to be potent cytotoxins. Our short semisynthesis and analogue-oriented synthesis approaches provide a series of lissoclimide natural products and analogues that expand the structure-activity relationships (SARs) in this family. The semisynthesis approach yielded significant quantities of chlorolissoclimide (CL) to permit an evaluation against the National Cancer Institute's 60-cell line panel and allowed us to obtain an X-ray co-crystal structure of the synthetic secondary metabolite with the eukaryotic 80S ribosome. Although it shares a binding site with other imide-based natural product translation inhibitors, CL engages in a particularly interesting and novel face-on halogen-π interaction between the ligand's alkyl chloride and a guanine residue. Our analogue-oriented synthesis provides many more lissoclimide compounds, which were tested against aggressive human cancer cell lines and for protein synthesis inhibitory activity. Finally, computational modelling was used to explain the SARs of certain key compounds and set the stage for the structure-guided design of better translation inhibitors.
Assuntos
Diterpenos/síntese química , Diterpenos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Succinimidas/síntese química , Succinimidas/farmacologia , Animais , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Diterpenos/química , Ensaios de Seleção de Medicamentos Antitumorais , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Peptídeos Cíclicos , Succinimidas/químicaRESUMO
Protein synthesis plays an essential role in cell proliferation, differentiation, and survival. Inhibitors of eukaryotic translation have entered the clinic, establishing the translation machinery as a promising target for chemotherapy. A recently discovered, structurally unique marine sponge-derived brominated alkaloid, (-)-agelastatin A (AglA), possesses potent antitumor activity. Its underlying mechanism of action, however, has remained unknown. Using a systematic top-down approach, we show that AglA selectively inhibits protein synthesis. Using a high-throughput chemical footprinting method, we mapped the AglA-binding site to the ribosomal A site. A 3.5 Å crystal structure of the 80S eukaryotic ribosome from S. cerevisiae in complex with AglA was obtained, revealing multiple conformational changes of the nucleotide bases in the ribosome accompanying the binding of AglA. Together, these results have unraveled the mechanism of inhibition of eukaryotic translation by AglA at atomic level, paving the way for future structural modifications to develop AglA analogs into novel anticancer agents.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Oxazolidinonas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Alcaloides/metabolismo , Antineoplásicos/metabolismo , Produtos Biológicos/metabolismo , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Oxazolidinonas/metabolismo , Conformação Proteica , Ribossomos/efeitos dos fármacos , Ribossomos/genéticaRESUMO
Cardiovascular diseases have been associated with genetic variants and increased plasma level of the secreted protein PCSK9. In this issue of Cell Chemical Biology, Petersen et al. (2016) describe an inhibitor of PCSK9 secretion in human cells that, surprisingly, targets the 80S ribosome.
Assuntos
Doenças Cardiovasculares , Eucariotos , Humanos , Pró-Proteína Convertase 9 , Biossíntese de Proteínas , Ribossomos , SecretagogosRESUMO
DR0248 is a protein identified in the Deinococcus radiodurans (DR) genome that is predicted to encompass two domains: an N-terminal minimal nucleotidyl transferase domain (MNT) and a C-terminal higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN). These two domains, usually encoded in two ORFs, have been suggested to play the role of a toxin-antitoxin (TA) system in prokaryotes. Recombinant DR0248 was overexpressed and purified from Escherichia coli and diffraction-quality crystals were obtained in the presence of the detergent molecules dodecyldimethylamine oxide (DDAO) and octaethylene glycol monododecyl ether (C12E8), which were used as crystallization additives. Crystals grown with DDAO diffracted to a resolution of 2.24â Å and belonged to space group C222(1), with unit-cell parameters a=98.4, b=129.9, c=59.2â Å. Crystals grown with C12E8 diffracted to a resolution of 1.83â Å and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=51.6, b=87.2, c=108.2â Å. The structure was solved by multiwavelength anomalous dispersion from zinc bound to the protein using a single crystal obtained in the presence of DDAO.
Assuntos
Proteínas de Bactérias/química , Deinococcus , Nucleotidiltransferases/química , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Cromatografia em Gel , Cristalização , Cristalografia por Raios X , Nucleotidiltransferases/isolamento & purificaçãoRESUMO
Repair of DNA double-strand breaks (DSBs) is essential for cell survival and maintaining genome integrity. DSBs are repaired in a stepwise manner by homologous recombination. Here, we focused on the early steps of DSB repair, including DSB recognition, which is still only poorly understood. In prokaryotes, this process has been proposed to involve the RecN protein, a member of the structural maintenance of chromosome (SMC) protein family, which include key eukaryotic and prokaryotic proteins such as cohesin, condensin, and Rad50. An extensive high- and low-resolution structural analysis of Deinococcus radiodurans RecN using a combination of protein crystallography and small-angle X-ray scattering enabled us to assemble a quasi-atomic model of the entire RecN protein, representing the complete structure of a SMC-like protein. These results, together with a thorough biochemical and mutational study of RecN, allow us to propose a model for the role of RecN in DSB repair.