RESUMO
The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.
RESUMO
Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.
Assuntos
Adaptação Fisiológica , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Fluorescência , Simulação de Acoplamento Molecular , Membrana Nuclear/metabolismo , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Domínios Proteicos , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Aminoacil-tRNA Sintetases/metabolismo , Antituberculosos/farmacologia , Teorema de Bayes , Evolução Biológica , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
The γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human γ-TuRC at â¼3.8 Å resolution, revealing an asymmetric, cone-shaped structure. Pseudo-atomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the γ-TuRC "seam." We also identify an unanticipated structural bridge that includes an actin-like protein and spans the γ-TuRC lumen. Despite its asymmetric architecture, the γ-TuRC arranges γ-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the γ-TuRC subunits introduces large (>100,000 Å2) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the γ-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.
Assuntos
Centro Organizador dos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/ultraestrutura , Tubulina (Proteína)/ultraestrutura , Actinas/metabolismo , Microscopia Crioeletrônica/métodos , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.
Assuntos
Metiltransferases/química , RNA Helicases/química , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Replicação Viral , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , RNA-Polimerase RNA-Dependente de Coronavírus , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Magnésio/metabolismo , Metiltransferases/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , RNA Viral/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2 , Proteínas não Estruturais Virais/metabolismoRESUMO
The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Leveduras/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Fúngicas , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , RNA Mensageiro , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5' cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here, we determine high-resolution cryoelectron microscopy (cryo-EM) structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.
Assuntos
COVID-19 , Nucleotidiltransferases , Humanos , Nucleotidiltransferases/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Microscopia Crioeletrônica , RNA Viral/genéticaRESUMO
Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from cryogenic electron microscopy and AlphaFold2 models. Key features of the inner and outer rings were integrated into a comprehensive model. We resolved flexible connectors that tie together the core scaffold, along with equatorial transmembrane complexes and a lumenal ring that anchor this channel within the pore membrane. The organization of the nuclear double outer ring reveals an architecture that may be shared with ancestral NPCs. Additional connections between the core scaffold and the central transporter suggest that under certain conditions, a degree of local organization is present at the periphery of the transport machinery. These connectors may couple conformational changes in the scaffold to the central transporter to modulate transport. Collectively, this analysis provides insights into assembly, transport, and NPC evolution.
Assuntos
Poro Nuclear , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Membrana TransportadorasRESUMO
The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus , Microscopia Crioeletrônica , SARS-CoV-2 , Humanos , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/ultraestrutura , COVID-19/virologia , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , RNA Viral/biossíntese , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Especificidade por Substrato , Guanosina Trifosfato/metabolismo , Capuzes de RNARESUMO
Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an â¼50 MDa assembly comprised of â¼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin reuptake inhibitors (SSRIs). We show that SMARCA3, a chromatin-remodeling factor, is a target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer. Formation of this complex increases the DNA-binding affinity of SMARCA3 and its localization to the nuclear matrix fraction. In the dentate gyrus, both p11 and SMARCA3 are highly enriched in hilar mossy cells and basket cells. The SSRI fluoxetine induces expression of p11 in both cell types and increases the amount of the ternary complex of p11/annexin A2/SMARCA3. SSRI-induced neurogenesis and behavioral responses are abolished by constitutive knockout of SMARCA3. Our studies indicate a central role for a chromatin-remodeling factor in the SSRI/p11 signaling pathway and suggest an approach to the development of improved antidepressant therapies. PAPERCLIP:
Assuntos
Anexina A2/metabolismo , Proteínas de Ligação a DNA/metabolismo , Giro Denteado/metabolismo , Proteínas S100/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Feminino , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Fibras Musgosas Hipocampais/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Fatores de Transcrição/química , Difração de Raios XRESUMO
LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , Proteoma/análise , Ribonucleoproteínas/análise , Sequência de Aminoácidos , Animais , Regulação para Baixo , Genoma Humano , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Fases de Leitura Aberta , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Helicases , Ribonucleoproteínas/isolamento & purificação , Alinhamento de Sequência , Transativadores/química , Transativadores/isolamento & purificação , Transativadores/metabolismoRESUMO
Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure. We use single-particle cryoelectron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Iniciação da Transcrição Genética , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Conformação de Ácido Nucleico , Ligação Proteica/genética , Conformação ProteicaRESUMO
The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells1,2. Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide-major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction.
Assuntos
Centro Germinativo/citologia , Centro Germinativo/imunologia , Células T Auxiliares Foliculares/citologia , Células T Auxiliares Foliculares/imunologia , Animais , Proliferação de Células , Células Clonais/citologia , Células Clonais/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Masculino , Camundongos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Células T Auxiliares Foliculares/metabolismoRESUMO
Mass spectrometry (MS) is rapidly becoming an essential tool for biologists and biochemists in their efforts to throw light on molecular mechanisms within cellular systems. Used in unison with genome sequence data, MS has developed into the method of choice for identifying proteins, elucidating their posttranslational modifications, and reading out their functional interactions. Variations of the method have even begun to enable accurate mass determination of intact protein complexes, allowing for direct determination of subunit stoichiometry and the interactions between the subunits. Advances in mass spectrometric technologies have also led to great improvements in our ability to probe and define many of the other key molecular players in cells, including the all important lipid components. We provide here some perspectives on the explosion of applications of MS to protein science, systems biology, proteomics, lipidomics, and cell biology in general.
Assuntos
Espectrometria de Massas/métodos , Proteínas/análise , Células/química , Proteômica/métodosRESUMO
Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Switching de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Proteínas Supressoras de Tumor/genética , Animais , Linfócitos B , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/genética , Elementos Facilitadores Genéticos , Rearranjo Gênico , Humanos , Domínios MYND , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Hipermutação Somática de Imunoglobulina/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Single domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, as well as therapeutic and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research - i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat IgG. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.
RESUMO
Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits. By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide-binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.
Assuntos
Complexo Shelterina , Humanos , Espectrometria de Massas , Microscopia Eletrônica , Domínios Proteicos , Complexo Shelterina/químicaRESUMO
COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.
Assuntos
Anticorpos Neutralizantes , SARS-CoV-2 , Anticorpos de Domínio Único , Animais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Regiões Determinantes de Complementaridade , Saccharomyces cerevisiae/genética , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/genética , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic "scout fragments", which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.