RESUMO
The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.
Assuntos
DNA Mitocondrial , Proteínas Mitocondriais , Animais , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Humanos , Mamíferos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA/metabolismo , RNA Mitocondrial , RNA Citoplasmático Pequeno , Partícula de Reconhecimento de Sinal , Transcrição GênicaRESUMO
The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , COVID-19/genética , Anticorpos Antivirais , Polimorfismo Genético , Anticorpos Neutralizantes , Células GerminativasRESUMO
Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, replicates using seven essential proteins encoded by its genome. Among these, the UL30 DNA polymerase, complexed with the UL42 processivity factor, orchestrates leading and lagging strand replication of the 152 kb viral genome. UL30 polymerase is a prime target for antiviral therapy, and resistance to current drugs can arise in immunocompromised individuals. Using electron cryo-microscopy (cryo-EM), we unveil the dynamic changes of the UL30/UL42 complex with DNA in three distinct states. First, a pre-translocation state with an open fingers domain ready for nucleotide incorporation. Second, a halted elongation state where the fingers close, trapping dATP in the dNTP pocket. Third, a DNA-editing state involving significant conformational changes to allow DNA realignment for exonuclease activity. Additionally, the flexible UL30 C-terminal domain interacts with UL42, forming an extended positively charged surface binding to DNA, thereby enhancing processive synthesis. These findings highlight substantial structural shifts in the polymerase and its DNA interactions during replication, offering insights for future antiviral drug development.
Assuntos
Microscopia Crioeletrônica , DNA Viral , DNA Polimerase Dirigida por DNA , Herpesvirus Humano 1 , Proteínas Virais , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Proteínas Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/ultraestrutura , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , DNA Viral/metabolismo , DNA Viral/biossíntese , Replicação do DNA , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Replicação Viral , Ligação Proteica , ExodesoxirribonucleasesRESUMO
Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Ligação a RNA , Proteínas de Ligação a DNA/química , Espectrometria de MassasRESUMO
Mitochondrial polycistronic transcripts are extensively processed to give rise to functional mRNAs, rRNAs and tRNAs; starting with the release of tRNA elements through 5'-processing by RNase P (MRPP1/2/3-complex) and 3'-processing by RNase Z (ELAC2). Here, we show using in vitro experiments that MRPP1/2 is not only a component of the mitochondrial RNase P but that it retains the tRNA product from the 5'-processing step and significantly enhances the efficiency of ELAC2-catalyzed 3'-processing for 17 of the 22 tRNAs encoded in the human mitochondrial genome. Furthermore, MRPP1/2 retains the tRNA product after ELAC2 processing and presents the nascent tRNA to the mitochondrial CCA-adding enzyme. Thus, in addition to being an essential component of the RNase P reaction, MRPP1/2 serves as a processing platform for several down-stream tRNA maturation steps in human mitochondria. These findings are of fundamental importance for our molecular understanding of disease-related mutations in MRPP1/2, ELAC2 and mitochondrial tRNA genes.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Metiltransferases/metabolismo , Mitocôndrias/genética , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Humanos , Mitocôndrias/enzimologia , Proteínas de Neoplasias/metabolismoRESUMO
Mitochondrial RNA polymerase produces long polycistronic precursors that contain the mRNAs, rRNAs and tRNAs needed for mitochondrial translation. Mitochondrial RNase P (mt-RNase P) initiates the maturation of the precursors by cleaving at the 5' ends of the tRNAs. Human mt-RNase P is only active as a tripartite complex (mitochondrial RNase P proteins 1-3; MRPP1-3), whereas plant and trypanosomal RNase Ps (PRORPs)-albeit homologous to MRPP3-are active as single proteins. The reason for this discrepancy has so far remained obscure. Here, we present the crystal structure of human MRPP3, which features a remarkably distorted and hence non-productive active site that we propose will switch to a fully productive state only upon association with MRPP1, MRPP2 and pre-tRNA substrate. We suggest a mechanism in which MRPP1 and MRPP2 both deliver the pre-tRNA substrate and activate MRPP3 through an induced-fit process.
Assuntos
Ribonuclease P/química , Proteínas de Arabidopsis/química , Domínio Catalítico , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Subunidades Proteicas/químicaRESUMO
Polyadenylation, performed by poly(A) polymerases (PAPs), is a ubiquitous post-transcriptional modification that plays key roles in multiple aspects of RNA metabolism. Although cytoplasmic and nuclear PAPs have been studied extensively, the mechanism by which mitochondrial PAP (mtPAP) selects adenosine triphosphate over other nucleotides is unknown. Furthermore, mtPAP is unique because it acts as a dimer. However, mtPAP's dimerization requirement remains enigmatic. Here, we show the structural basis for mtPAP's nucleotide selectivity, dimerization and catalysis. Our structures reveal an intricate dimerization interface that features an RNA-recognition module formed through strand complementation. Further, we propose the structural basis for the N478D mutation that drastically reduces the length of poly(A) tails on mitochondrial mRNAs in patients with spastic ataxia 4 (SPAX4), a severe and progressive neurodegenerative disease.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas Mitocondriais/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Galinhas , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Humanos , Deficiência Intelectual/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Espasticidade Muscular/genética , Mutação , Nucleotídeos/química , Nucleotídeos/metabolismo , Nucleotidiltransferases/química , Atrofia Óptica/genética , Fenótipo , RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Ataxias Espinocerebelares/genéticaRESUMO
A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery.
Assuntos
DNA Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , 8-Hidroxi-2'-Desoxiguanosina , Sistema Livre de Células , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/genética , Desoxiguanosina/metabolismo , Genoma Mitocondrial/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Modelos Genéticos , Ligação Proteica , Proteínas Recombinantes/metabolismo , Moldes Genéticos , Fatores de Transcrição/genéticaRESUMO
Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (Tfam and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with Tfam to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of Tfam, both from promoters and non-specific DNA sequences. Additionally, when in presence of Tfam and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which Tfam specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Iniciação da Transcrição Genética , Animais , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Metiltransferases/metabolismo , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Mutação , Estrutura Terciária de Proteína , Fatores de Transcrição/metabolismoRESUMO
Proteins crucial for the respiratory chain are translated by the mitochondrial ribosome. Mitochondrial ribosome biogenesis is therefore critical for oxidative phosphorylation capacity and disturbances are known to cause human disease. This complex process is evolutionary conserved and involves several RNA processing and modification steps required for correct ribosomal RNA maturation. We recently showed that a member of the mitochondrial transcription termination factor (MTERF) family of proteins, MTERF4, recruits NSUN4, a 5-methylcytosine RNA methyltransferase, to the large ribosomal subunit in a process crucial for mitochondrial ribosome biogenesis. Here, we describe the 3D crystal structure of the human MTERF4-NSUN4 complex determined to 2.9 Å resolution. MTERF4 is composed of structurally repeated MTERF-motifs that form a nucleic acid binding domain. NSUN4 lacks an N- or C-terminal extension that is commonly used for RNA recognition by related RNA methyltransferases. Instead, NSUN4 binds to the C-terminus of MTERF4. A positively charged surface forms an RNA binding path from the concave to the convex side of MTERF4 and further along NSUN4 all of the way into the active site. This finding suggests that both subunits of the protein complex likely contribute to RNA recognition. The interface between MTERF4 and NSUN4 contains evolutionarily conserved polar and hydrophobic amino acids, and mutations that change these residues completely disrupt complex formation. This study provides a molecular explanation for MTERF4-dependent recruitment of NSUN4 to ribosomal RNA and suggests a unique mechanism by which other members of the large MTERF-family of proteins can regulate ribosomal biogenesis.
Assuntos
Proteínas de Transporte/química , Metiltransferases/química , Mitocôndrias/metabolismo , Ribossomos/química , Fatores de Transcrição/química , Domínio Catalítico , Biologia Computacional/métodos , Cristalografia por Raios X/métodos , Análise Mutacional de DNA , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Estrutura Terciária de Proteína , RNA Ribossômico/química , Proteínas Recombinantes/químicaRESUMO
Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+ß subunits) that cleaves the glucosyl-aglycone C-C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2ß2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C-C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min-1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min-1).
Assuntos
Proteínas de Bactérias , Deinococcus , Flavonoides , Genes Bacterianos , Família Multigênica , Xantonas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Deinococcus/genética , Deinococcus/metabolismo , Flavonoides/metabolismo , Flavonoides/química , Glicosídeos/metabolismo , Glicosídeos/química , Glicosilação , Modelos Moleculares , Xantonas/metabolismo , Xantonas/químicaRESUMO
Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.
Assuntos
Proteínas de Ligação ao GTP , Mitocôndrias , Ribossomos Mitocondriais , Fosforilação Oxidativa , Humanos , Ribossomos Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Células HEK293 , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Células HeLaRESUMO
Descendants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant now account for almost all SARS-CoV-2 infections. The Omicron variant and its sublineages have spike glycoproteins that are highly diverged from the pandemic founder and first-generation vaccine strain, resulting in significant evasion from monoclonal antibody therapeutics and vaccines. Understanding how commonly elicited antibodies can broaden to cross-neutralize escape variants is crucial. We isolate IGHV3-53, using "public" monoclonal antibodies (mAbs) from an individual 7 months post infection with the ancestral virus and identify antibodies that exhibit potent and broad cross-neutralization, extending to the BA.1, BA.2, and BA.4/BA.5 sublineages of Omicron. Deep mutational scanning reveals these mAbs' high resistance to viral escape. Structural analysis via cryoelectron microscopy of a representative broadly neutralizing antibody, CAB-A17, in complex with the Omicron BA.1 spike highlights the structural underpinnings of this broad neutralization. By reintroducing somatic hypermutations into a germline-reverted CAB-A17, we delineate the role of affinity maturation in the development of cross-neutralization by a public class of antibodies.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2/imunologia , Humanos , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Reações Cruzadas/imunologia , Microscopia Crioeletrônica , Testes de NeutralizaçãoRESUMO
The continued evolution of SARS-CoV-2 underscores the need to understand qualitative aspects of the humoral immune response elicited by spike immunization. Here, we combine monoclonal antibody (mAb) isolation with deep B cell receptor (BCR) repertoire sequencing of rhesus macaques immunized with prefusion-stabilized spike glycoprotein. Longitudinal tracing of spike-sorted B cell lineages in multiple immune compartments demonstrates increasing somatic hypermutation and broad dissemination of vaccine-elicited B cells in draining and non-draining lymphoid compartments, including the bone marrow, spleen and, most notably, periaortic lymph nodes. Phylogenetic analysis of spike-specific monoclonal antibody lineages identified through deep repertoire sequencing delineates extensive intra-clonal diversification that shaped neutralizing activity. Structural analysis of the spike in complex with a broadly neutralizing mAb provides a molecular basis for the observed differences in neutralization breadth between clonally related antibodies. Our findings highlight that immunization leads to extensive intra-clonal B cell evolution where members of the same lineage can both retain the original epitope specificity and evolve to recognize additional spike variants not previously encountered.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos B , Macaca mulatta , Filogenia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Linfócitos B/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , COVID-19/imunologia , COVID-19/virologia , Humanos , Vacinas contra COVID-19/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , ImunizaçãoRESUMO
How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers and undergoes liquid-liquid phase separation by binding RNA or ribosomal proteins. It provides the scaffold for ribosome assembly but also prevents protein aggregation as part of the cellular stress response. Here, we use aggregation assays and native mass spectrometry (MS) to examine the relationship between the self-assembly and chaperone activity of NPM1. We find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning-based structure prediction and cryo-electron microscopy reveal fuzzy interactions between the acidic disordered region and the C-terminal nucleotide-binding domain, which cross-link NPM1 pentamers into partially disordered oligomers. The addition of basic peptides results in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. Together, our findings show that NPM1 uses a "grappling hook" mechanism to form a network-like structure that traps aggregation-prone proteins. Nucleolar proteins and RNAs simultaneously modulate the association strength and chaperone activity, suggesting a mechanism by which nucleolar composition regulates the chaperone activity of NPM1.
RESUMO
Perturbed cell adhesion mechanisms are crucial for tumor invasion and metastasis. A cell adhesion protein, TSLC1 (tumor suppressor in lung cancer 1), is inactivated in a majority of metastatic cancers. DAL-1 (differentially expressed in adenocarcinoma of the lung protein), another tumor suppressor, binds through its FERM domain to the TSLC1 C-terminal, 4.1 glycophorin C-like, cytoplasmic domain. However, the molecular basis for this interaction is unknown. Here, we describe the crystal structure of a complex between the DAL-1 FERM domain and a portion of the TSLC1 cytoplasmic domain. DAL-1 binds to TSLC1 through conserved residues in a well defined hydrophobic pocket in the structural C-lobe of the DAL-1 FERM domain. From the crystal structure, it is apparent that Tyr(406) and Thr(408) in the TSLC1 cytoplasmic domain form the most important interactions with DAL-1, and this was also confirmed by surface plasmon resonance studies. Our results refute earlier exon deletion experiments that indicated that glycophorin C interacts with the α-lobe of 4.1 FERM domains.
Assuntos
Moléculas de Adesão Celular/química , Imunoglobulinas/química , Proteínas de Membrana/química , Proteínas Supressoras de Tumor/química , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cristalografia por Raios X , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Biespecíficos/metabolismo , COVID-19/virologia , Chlorocebus aethiops , Microscopia Crioeletrônica , Células HEK293 , Humanos , Camundongos Transgênicos , Testes de Neutralização/métodos , Ligação Proteica , Conformação Proteica , Multimerização Proteica/imunologia , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
Assuntos
Fracionamento Químico/métodos , Físico-Química/métodos , Engenharia de Proteínas/métodos , Proteômica/métodos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Mitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation.
Assuntos
Ribossomos Mitocondriais/química , Subunidades Ribossômicas Maiores/química , Linhagem Celular , Microscopia Crioeletrônica , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Ligação Proteica , Dobramento de RNA , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.