RESUMO
Nucleic acid processing enzymes use a two-Mg2+-ion motif to promote the formation and cleavage of phosphodiester bonds. Yet, recent evidence demonstrates the presence of spatially conserved second-shell cations surrounding the catalytic architecture of proteinaceous and RNA-dependent enzymes. The RNase mitochondrial RNA processing (MRP) complex, which cleaves the ribosomal RNA (rRNA) precursor at the A3 cleavage site to yield mature 5'-end of 5.8S rRNA, hosts in the catalytic core one atypically-located Mg2+ ion, in addition to the ions forming the canonical catalytic motif. Here, we employ biased quantum classical molecular dynamics simulations of RNase MRP to discover that the third Mg2+ ion inhibits the catalytic process. Instead, its displacement in favour of a second-shell monovalent K+ ion propels phosphodiester bond cleavage by enabling the formation of a specific hydrogen bonding network that mediates the essential proton transfer step. This study points to a direct involvement of a transient K+ ion in the catalytic cleavage of the phosphodiester bond and implicates cation trafficking as a general mechanism in nucleic acid processing enzymes and ribozymes.
RESUMO
Cleavage and formation of phosphodiester bonds in nucleic acids is accomplished by large cellular machineries composed of both protein and RNA. Long thought to rely on a two-metal-ion mechanism for catalysis, structure comparisons revealed many contain highly spatially conserved second-shell monovalent cations, whose precise function remains elusive. A recent high-resolution structure of the spliceosome, essential for pre-mRNA splicing in eukaryotes, revealed a potassium ion in the active site. Here, we employ biased quantum mechanics/ molecular mechanics molecular dynamics to elucidate the function of this monovalent ion in splicing. We discover that the K+ ion regulates the kinetics and thermodynamics of the first splicing step by rigidifying the active site and stabilizing the substrate in the pre- and post-catalytic state via formation of key hydrogen bonds. Our work supports a direct role for the K+ ion during catalysis and provides a mechanistic hypothesis likely shared by other nucleic acid processing enzymes.
Assuntos
RNA , Spliceossomos , Spliceossomos/metabolismo , RNA/metabolismo , Splicing de RNA , Catálise , Metais/metabolismo , Potássio/metabolismo , Quelantes/metabolismo , Conformação de Ácido Nucleico , Sítios de Ligação , Cátions Monovalentes/metabolismoRESUMO
Natural killer (NK) cells play an important role in the innate immune response against tumors and various pathogens such as viruses and bacteria. Their function is controlled by a wide array of activating and inhibitory receptors, which are expressed on their cell surface. Among them is a dimeric NKG2A/CD94 inhibitory transmembrane (TM) receptor which specifically binds to the non-classical MHC I molecule HLA-E, which is often overexpressed on the surface of senescent and tumor cells. Using the Alphafold 2 artificial intelligence system, we constructed the missing segments of the NKG2A/CD94 receptor and generated its complete 3D structure comprising extracellular (EC), TM, and intracellular regions, which served as a starting point for the multi-microsecond all-atom molecular dynamics simulations of the receptor with and without the bound HLA-E ligand and its nonameric peptide. The simulated models revealed that an intricate interplay of events is taking place between the EC and TM regions ultimately affecting the intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) regions that host the point at which the signal is transmitted further down the inhibitory signaling cascade. Signal transduction through the lipid bilayer was also coupled with the changes in the relative orientation of the NKG2A/CD94 TM helices in response to linker reorganization, mediated by fine-tuned interactions in the EC region of the receptor, taking place after HLA-E binding. This research provides atomistic details of the cells' protection mechanism against NK cells and broadens the knowledge regarding the TM signaling of ITIM-bearing receptors.
Assuntos
Subfamília C de Receptores Semelhantes a Lectina de Células NK , Receptores Imunológicos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores de Células Matadoras Naturais/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Ligantes , Inteligência Artificial , Antígenos de Histocompatibilidade Classe I/metabolismo , Transdução de Sinais , Proteínas de Transporte/metabolismo , Antígenos HLA-ERESUMO
MHC class I antigen E (HLA-E), a ligand for the inhibitory NKG2A/CD94 receptor of the immune system, is responsible for evading the immune surveillance in several settings, including senescent cell accumulation and tumor persistence. The formation of this ligand-receptor interaction promotes the inhibition of the cytolytic action of immune system natural killer (NK) cells and CD8+ T-cells expressing this receptor. The final outcome of the HLA-E/NKG2A/CD94 interaction on target cells is also highly dependent on the identity of the nonameric peptide incorporated into the HLA-E ligand. To better understand the role played by a nonameric peptide in these immune complexes, we performed a series of multi-microsecond all-atom molecular dynamics simulations. We generated natural and alternative variants of the nonameric peptide bound to the HLA-E ligand alone or in the HLA-E/NKG2A/CD94 complexes. A systematic study of molecular recognition between HLA-E and peptides led to the development of new variants that differ at the strategic 6th position (P6) of the peptide and have favorable in silico properties comparable to those of natural binding peptides. Further examination of a selected subset of peptides in full complexes revealed a new variant that, according to our previously derived atomistic model, can interfere with the signal transduction via HLA-E/NKG2A/CD94 and thus prevent the target cell from evading immune clearance by NK and CD8+ T-cells. These simulations provide an atomistic picture of how a small change in amino acid sequence can lead to a profound effect on binding and molecular recognition. Furthermore, our study also provides new data on the peptide interaction motifs as well as the energetic and conformational properties of the binding interface, laying the structure-based foundation for future development of potential therapeutic peptides, peptidomimetics, or even small molecules that would bind to the HLA-E ligand and abrogate NKG2A/CD94 recognition. Such external intervention would be useful in the emerging field of targeting senescent cells in a variety of age-related diseases, as well as in novel cancer immunotherapies.
RESUMO
Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.
Assuntos
Oomicetos , Lipídeos , Necrose , Oomicetos/metabolismo , Perforina/metabolismo , Plantas/metabolismo , Proteínas/metabolismoRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents the most severe global health crisis in modern human history. One of the major SARS-CoV-2 virulence factors is nonstructural protein 1 (Nsp1), which, outcompeting with the binding of host mRNA to the human ribosome, triggers a translation shutdown of the host immune system. Here, microsecond-long all-atom simulations of the C-terminal portion of the SARS-CoV-2/SARS-CoV Nsp1 in complex with the 40S ribosome disclose that SARS-CoV-2 Nsp1 has evolved from its SARS-CoV ortholog to more effectively hijack the ribosome by undergoing a critical switch of Q/E158 and E/Q159 residues that perfects Nsp1's interactions with the ribosome. Our outcomes offer a basis for understanding the sophisticated mechanisms underlying SARS-CoV-2 diversion and exploitation of human cell components to its deadly purposes.
Assuntos
Simulação de Dinâmica Molecular , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Humanos , Ligação de Hidrogênio , Ligação Proteica , Subunidades Ribossômicas Menores de Eucariotos/química , SARS-CoV-2/isolamento & purificação , Proteínas não Estruturais Virais/químicaRESUMO
The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre-mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B-8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B-8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome-mutant cancer cells remains elusive. Here, microsecond-long all-atom simulations addressed the binding/dissociation mechanism of H3B-8800 to wild type and K700E SF3B1-containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B-8800 binding. This supports the hypothesis that the selectivity of H3B-8800 towards mutant cancer cells is unrelated to its preferential targeting of K700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B-8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug-discovery efforts to specifically target mutant SF3b.
Assuntos
Mutação , Neoplasias/patologia , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Piperazinas/metabolismo , Piridinas/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Humanos , Simulação de Dinâmica Molecular , Neoplasias/genética , Neoplasias/metabolismo , Fenótipo , Fosfoproteínas/genética , Piperazinas/química , Conformação Proteica , Piridinas/química , Fatores de Processamento de RNA/genéticaRESUMO
Natural killer (NK) cells, an important part of the innate immune system, can clear a wide variety of pathological challenges, including tumor, senescent, and virally infected cells. They express various activating and inhibitory receptors on their surface, and the balance of interactions between them and specific ligands displayed on the surface of target cells is critical for NK cell cytolytic function and target cell protection. The CD94/NKG2A heterodimer is one of the inhibitory receptors that interacts with its trimeric ligand consisting of HLA-E, ß2m, and a nonameric peptide. Here, multi-microsecond-long all-atom molecular dynamics simulations of eight immune complexes elucidate the subtleties of receptor (NKG2A/CD94)-ligand (HLA-E/ß2m/peptide) molecular recognition that mediate the NK cell protection from a geometric and energetic perspective. We identify key differences in the interactions between the receptor and ligand complexes, which are via an entangled network of hydrogen bonds fine-tuned by the ligand-specific nonameric peptide. We further reveal that the receptor protein NKG2A regulates the NK cell activity, while its CD94 partner forms the majority of the energetically important interactions with the ligand. This knowledge rationalizes the atomistic details of the fundamental NK cell protection mechanism and may enable a variety of opportunities in rational-based drug discovery for diverse pathologies including viral infections and cancer and elimination of senescent cells associated with potential treatment of many age-related diseases.
Assuntos
Complexo Antígeno-Anticorpo , Antígenos de Histocompatibilidade Classe I , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Humanos , Células Matadoras Naturais , Peptídeos , Antígenos HLA-ERESUMO
The innate immune system's natural killer (NK) cells exert their cytolytic function against a variety of pathological challenges, including tumors and virally infected cells. Their activation depends on net signaling mediated via inhibitory and activating receptors that interact with specific ligands displayed on the surfaces of target cells. The CD94/NKG2C heterodimer is one of the NK activating receptors and performs its function by interacting with the trimeric ligand comprised of the HLA-E/ß2m/nonameric peptide complex. Here, simulations of the all-atom multi-microsecond molecular dynamics in five immune complexes provide atomistic insights into the receptor-ligand molecular recognition, as well as the molecular events that facilitate the NK cell activation. We identify NKG2C, the HLA-Eα2 domain, and the nonameric peptide as the key elements involved in the molecular machinery of signal transduction via an intertwined hydrogen bond network. Overall, the study addresses the complex intricacies that are necessary to understand the mechanisms of the innate immune system.
Assuntos
Complexo Antígeno-Anticorpo/química , Antígenos de Histocompatibilidade Classe I/química , Modelos Moleculares , Subfamília C de Receptores Semelhantes a Lectina de Células NK/química , Subfamília D de Receptores Semelhantes a Lectina de Células NK/química , Peptídeos/química , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ligação de Hidrogênio , Ligantes , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Relação Estrutura-Atividade , Antígenos HLA-ERESUMO
The rapid and relentless emergence of novel highly transmissible SARS-CoV-2 variants, possibly decreasing vaccine efficacy, currently represents a formidable medical and societal challenge. These variants frequently hold mutations on the Spike protein's receptor-binding domain (RBD), which, binding to the angiotensin-converting enzyme 2 (ACE2) receptor, mediates viral entry into host cells. Here, all-atom molecular dynamics simulations and dynamical network theory of the wild-type and mutant RBD/ACE2 adducts disclose that while the N501Y mutation (UK variant) enhances the Spike's binding affinity toward ACE2, the concomitant N501Y, E484K, and K417N mutations (South African variant) aptly adapt to increase SARS-CoV-2 propagation via a two-pronged strategy: (i) effectively grasping ACE2 through an allosteric signaling between pivotal RBD structural elements and (ii) impairing the binding of antibodies elicited by infected or vaccinated patients. This information unlocks the molecular terms and evolutionary strategies underlying the increased virulence of emerging SARS-CoV-2 variants, setting the basis for developing the next-generation anti-COVID-19 therapeutics.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica/genética , Domínios Proteicos/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , TermodinâmicaRESUMO
The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.
Assuntos
Phytophthora/patogenicidade , Doenças das Plantas/prevenção & controle , Pythium/patogenicidade , Solanum tuberosum/genética , Simulação de Dinâmica Molecular , Necrose , Phytophthora/genética , Doenças das Plantas/parasitologia , Folhas de Planta/genética , Folhas de Planta/parasitologia , Pythium/genética , Solanum tuberosum/parasitologia , Ressonância de Plasmônio de Superfície , Nicotiana/genética , Nicotiana/parasitologiaRESUMO
Over one third of biomolecules rely on metal ions to exert their cellular functions. Metal ions can play a structural role by stabilizing the structure of biomolecules, a functional role by promoting a wide variety of biochemical reactions, and a regulatory role by acting as messengers upon binding to proteins regulating cellular metal-homeostasis. These diverse roles in biology ascribe critical implications to metal-binding proteins in the onset of many diseases. Hence, it is of utmost importance to exhaustively unlock the different mechanistic facets of metal-binding proteins and to harness this knowledge to rationally devise novel therapeutic strategies to prevent or cure pathological states associated with metal-dependent cellular dysfunctions. In this compendium, we illustrate how the use of a computational arsenal based on docking, classical, and quantum-classical molecular dynamics simulations can contribute to extricate the minutiae of the catalytic, transport, and inhibition mechanisms of metal-binding proteins at the atomic level. This knowledge represents a fertile ground and an essential prerequisite for selectively targeting metal-binding proteins with small-molecule inhibitors aiming to (i) abrogate deregulated metal-dependent (mis)functions or (ii) leverage metal-dyshomeostasis to selectively trigger harmful cells death.
Assuntos
Proteínas de Transporte/metabolismo , Metais Pesados/metabolismo , Proteínas de Transporte/química , Biologia Computacional , Metais Pesados/química , Modelos Moleculares , Conformação MolecularRESUMO
As the world's population progressively ages, the burden on the socio-economic and health systems is escalating, demanding sustainable and lasting solutions. Cellular senescence, one of the hallmarks of ageing, is a state of irreversible cell cycle arrest that occurs in response to various genotoxic stressors and is considered an important factor in the development of many age-related diseases and therefore a potential therapeutic target. Here, the role of senescent cells in age-related diseases is discussed, focusing on their formation and main characteristics. The mechanisms leading to senescent cells are presented, including replicative and premature senescence as well as senescence that occurs in various physiological processes, such as wound healing. The second part comprises a comprehensive description of various biomarkers currently used for the detection of senescent cells along with the investigated therapeutic approaches, namely senolytics, senomorphics and the clearance of senescent cells by the immune system. Potential delivery systems suitable for such therapies and model organisms to study senescence are also briefly examined. This in-depth overview of cellular senescence contributes to a deeper understanding of a rapidly evolving area aimed to tackle the age-related diseases in a more mechanistic way, as well as highlights future research opportunities.
Assuntos
Senescência Celular , Dano ao DNA , Biomarcadores , Sistema ImunitárioRESUMO
Intron removal from premature-mRNA (pre-mRNA splicing) is an essential part of gene expression and regulation that is required for the production of mature, protein-coding mRNA. The spliceosome (SPL), a majestic machine composed of five small nuclear RNAs and hundreds of proteins, behaves as an eminent transcriptome tailor, efficiently performing splicing as a protein-directed metallo-ribozyme. To select and excise long and diverse intronic sequences with single-nucleotide precision, the SPL undergoes a continuous compositional and conformational remodeling, forming eight distinct complexes throughout each splicing cycle. Splicing fidelity is of paramount importance to preserve the integrity of the proteome. Mutations in splicing factors can severely compromise the accuracy of this machinery, leading to aberrant splicing and altered gene expression. Decades of biochemical and genetic studies have provided insights into the SPL's composition and function, but its complexity and plasticity have prevented an in-depth mechanistic understanding. Single-particle cryogenic electron microscopy techniques have ushered in a new era for comprehending eukaryotic gene regulation, providing several near-atomic resolution structures of the SPL from yeast and humans. Nevertheless, these structures represent isolated snapshots of the splicing process and are insufficient to exhaustively assess the function of each SPL component and to unravel particular facets of the splicing mechanism in a dynamic environment.In this Account, building upon our contributions in this field, we discuss the role of biomolecular simulations in uncovering the mechanistic intricacies of eukaryotic splicing in health and disease. Specifically, we showcase previous applications to illustrate the role of atomic-level simulations in elucidating the function of specific proteins involved in the architectural reorganization of the SPL along the splicing cycle. Moreover, molecular dynamics applications have uniquely contributed to decrypting the channels of communication required for critical functional transitions of the SPL assemblies. They have also shed light on the role of carcinogenic mutations in the faithful selection of key intronic regions and the molecular mechanism of splicing modulators. Additionally, we emphasize the role of quantum-classical molecular dynamics in unraveling the chemical details of pre-mRNA cleavage in the SPL and in its evolutionary ancestors, group II intron ribozymes. We discuss methodological pitfalls of multiscale calculations currently used to dissect the splicing mechanism, presenting future challenges in this field. The results highlight how atomic-level simulations can enrich the interpretation of experimental results. We envision that the synergy between computational and experimental approaches will aid in developing innovative therapeutic strategies and revolutionary gene modulation tools to fight the over 200 human diseases associated with splicing misregulation, including cancer and neurodegeneration.
Assuntos
Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Antineoplásicos/farmacologia , Humanos , Simulação de Dinâmica Molecular , Neoplasias/genética , Neoplasias/patologia , Teoria Quântica , Splicing de RNA/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismoRESUMO
Proline-rich antimicrobial peptides (PrAMPs) are promising lead compounds for developing new antimicrobials; however, their narrow spectrum of action is limiting. PrAMPs kill bacteria binding to their ribosomes and inhibiting protein synthesis. In this study, 133 derivatives of the PrAMP Bac7(1-16) were synthesized to identify the crucial residues for ribosome inactivation and antimicrobial activity. Then, five new Bac7(1-16) derivatives were conceived and characterized by antibacterial and membrane permeabilization assays, X-ray crystallography, and molecular dynamics simulations. Some derivatives displayed broad spectrum activity, encompassing Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Staphylococcus aureus. Two peptides out of five acquired a weak membrane-perturbing activity while maintaining the ability to inhibit protein synthesis. These derivatives became independent of the SbmA transporter, commonly used by native PrAMPs, suggesting that they obtained a novel route to enter bacterial cells. PrAMP-derived compounds could become new-generation antimicrobials to combat antibiotic-resistant pathogens.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Prolina/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Testes de Sensibilidade Microbiana , Permeabilidade , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismoRESUMO
To achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.
Assuntos
Acetilglucosaminidase/metabolismo , Proteínas de Bactérias/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/enzimologia , Acetilglucosaminidase/química , Acetilglucosaminidase/genética , Regulação Alostérica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Hidrólise , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , Domínios Proteicos , Staphylococcus aureus/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Intron splicing of a nascent mRNA transcript by spliceosome (SPL) is a hallmark of gene regulation in eukaryotes. SPL is a majestic molecular machine composed of an entangled network of proteins and RNAs that meticulously promotes intron splicing through the formation of eight intermediate complexes. Cross-communication among the critical distal proteins of the SPL assembly is pivotal for fast and accurate directing of the compositional and conformational readjustments necessary to achieve high splicing fidelity. Here, molecular dynamics (MD) simulations of an 800â¯000 atom model of SPL C complex from yeast Saccharomyces cerevisiae and community network analysis enabled us to decrypt the complexity of this huge molecular machine, by identifying the key channels of information transfer across long distances separating key protein components. The reported study represents an unprecedented attempt in dissecting cross-communication pathways within one of the most complex machines of eukaryotic cells, supporting the critical role of Clf1 and Cwc2 splicing cofactors and specific domains of the Prp8 protein as signal conveyors for pre-mRNA maturation. Our findings provide fundamental advances into mechanistic aspects of SPL, providing a conceptual basis for controlling the SPL via small-molecule modulators able to tackle splicing-associated diseases by altering/obstructing information-exchange paths.
Assuntos
Spliceossomos/metabolismo , Íntrons , Simulação de Dinâmica Molecular , Análise de Componente Principal , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/química , Spliceossomos/química , Spliceossomos/genéticaRESUMO
Splicing modulators (SMs) pladienolides, herboxidienes, and spliceostatins exert their antitumor activity by altering the ability of SF3B1 and PHF5A proteins, components of SF3b splicing factor, to recognize distinct intron branching point sequences, thus finely calibrating constitutive/alternative/aberrant splicing of pre-mRNA. Here, by exploiting structural information obtained from cryo-EM data, and by performing multiple µs-long all-atom simulations of SF3b in apo form and in complex with selected SMs, we disclose how these latter seep into the narrow slit at the SF3B1/PHF5A protein interface. This locks the intrinsic open/closed conformational transitions of SFB1's solenoidal structure into the open state. As a result, SMs prevent the formation of a closed/intron-loaded conformation of the SF3B1 protein by decreasing the internal SF3B1 cross-correlation and reducing SF3B1's functional plasticity. We further compellingly support the proposition that SMs' action exceeds a purely competitive inhibition. Indeed, our simulations also demonstrate that the introduction of recurrent drug resistance/sensitizing mutations in SF3B1 or PHF5A, besides affecting the binding affinity of SMs, likewise influence the functional dynamics of SF3B1. This knowledge clarifies the molecular terms of SF3b modulation by small-molecules, fostering the rational-based discovery of drugs tackling distinct cancer types resulting from dysregulated splicing. This work also supports the coming of age usage of cryo-EM structural data in forthcoming drug-discovery studies.
Assuntos
Fosfoproteínas , Splicing de RNA , Microscopia Crioeletrônica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3'-splice-site (3'SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered BPS binding and 3'SS selection, lead to mis-spliced mRNA transcripts. Although these mutations recur in hematologic malignancies, the mechanism by which they change gene expression remains unclear. In this study, multi-microsecond-long molecular-dynamics simulations of eighth distinct â¼700,000 atom models of the spliceosome Bact complex, and gene sequencing of SF3B1, disclose that these carcinogenic isoforms destabilize intron binding and/or affect the functional dynamics of Hsh155/SF3B1 only when binding non-consensus BPSs, as opposed to the non-pathogenic variants newly annotated here. This pinpoints a cross-talk between the distal Hsh155 mutation and BPS recognition sites. Our outcomes unprecedentedly contribute to elucidating the principles of pre-mRNA recognition, which provides critical insights on the mechanism underlying constitutive/alternative/aberrant splicing.
Assuntos
Carcinógenos/química , Simulação de Dinâmica Molecular , Mutação , Fosfoproteínas/química , Fosfoproteínas/genética , Precursores de RNA/química , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Humanos , Análise de Componente Principal , Precursores de RNA/genética , Splicing de RNA/genética , Saccharomyces cerevisiae/genética , Eletricidade EstáticaRESUMO
Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic microorganisms. They trigger necrosis in various eudicot plants upon binding to plant sphingolipid glycosylinositol phosphorylceramides (GIPC). Interestingly, HaNLP3 from the obligate biotroph oomycete Hyaloperonospora arabidopsidis does not induce necrosis. We determined the crystal structure of HaNLP3 and showed that it adopts the NLP fold. However, the conformations of the loops surrounding the GIPC headgroup-binding cavity differ from those of cytotoxic Pythium aphanidermatum NLPPya. Essential dynamics extracted from µs-long molecular dynamics (MD) simulations reveals a limited conformational plasticity of the GIPC-binding cavity in HaNLP3 relative to toxic NLPs. This likely precludes HaNLP3 binding to GIPCs, which is the underlying reason for the lack of toxicity. This study reveals that mutations at key protein regions cause a switch between non-toxic and toxic phenotypes within the same protein scaffold. Altogether, these data provide evidence that protein flexibility is a distinguishing trait of toxic NLPs and highlight structural determinants for a potential functional diversification of non-toxic NLPs utilized by biotrophic plant pathogens.