RESUMEN
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Microscopía por Crioelectrón , ADN , Edición Génica , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , ADN/metabolismo , ADN/genética , Edición Génica/métodos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Células HEK293 , Dominios Proteicos , Genoma Humano , Modelos Moleculares , Estructura Terciaria de Proteína , Conformación de Ácido Nucleico , Biocatálisis , Magnesio/química , Magnesio/metabolismoRESUMEN
Cav1.2 channels play crucial roles in various neuronal and physiological processes. Here, we present cryo-EM structures of human Cav1.2, both in its apo form and in complex with several drugs, as well as the peptide neurotoxin calciseptine. Most structures, apo or bound to calciseptine, amlodipine, or a combination of amiodarone and sofosbuvir, exhibit a consistent inactivated conformation with a sealed gate, three up voltage-sensing domains (VSDs), and a down VSDII. Calciseptine sits on the shoulder of the pore domain, away from the permeation path. In contrast, when pinaverium bromide, an antispasmodic drug, is inserted into a cavity reminiscent of the IFM-binding site in Nav channels, a series of structural changes occur, including upward movement of VSDII coupled with dilation of the selectivity filter and its surrounding segments in repeat III. Meanwhile, S4-5III merges with S5III to become a single helix, resulting in a widened but still non-conductive intracellular gate.
Asunto(s)
Canales de Calcio Tipo L , Venenos Elapídicos , Humanos , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Neurotoxinas , Dominios Proteicos , Microscopía por CrioelectrónRESUMEN
The endoplasmic reticulum (ER) is the site of membrane protein insertion, folding, and assembly in eukaryotes. Over the past few years, a combination of genetic and biochemical studies have implicated an abundant factor termed the ER membrane protein complex (EMC) in several aspects of membrane protein biogenesis. This large nine-protein complex is built around a deeply conserved core formed by the EMC3-EMC6 subcomplex. EMC3 belongs to the universally conserved Oxa1 superfamily of membrane protein transporters, whereas EMC6 is an ancient, widely conserved obligate partner. EMC has an established role in the insertion of transmembrane domains (TMDs) and less understood roles during the later steps of membrane protein folding and assembly. Several recent structures suggest hypotheses about the mechanism(s) of TMD insertion by EMC, with various biochemical and proteomics studies beginning to reveal the range of EMC's membrane protein substrates.
Asunto(s)
Retículo Endoplásmico , Proteínas de la Membrana , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas , Dominios Proteicos , Pliegue de ProteínaRESUMEN
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.
Asunto(s)
Adaptación Fisiológica , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Fluorescencia , Simulación del Acoplamiento Molecular , Membrana Nuclear/metabolismo , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Dominios Proteicos , Reproducibilidad de los Resultados , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.
Asunto(s)
Enzima Convertidora de Angiotensina 2/química , Receptores Virales/química , SARS-CoV-2/química , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Mutación/genética , Filogenia , Unión Proteica , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Electricidad Estática , Homología Estructural de ProteínaRESUMEN
HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.
Asunto(s)
Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Virión/ultraestructura , Productos del Gen env del Virus de la Inmunodeficiencia Humana/ultraestructura , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/ultraestructura , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacología , Secuencia de Aminoácidos , Disulfuros/farmacología , Epítopos/química , Células HEK293 , Proteína gp41 de Envoltorio del VIH/química , Humanos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Modelos Moleculares , Pruebas de Neutralización , Péptidos/química , Polisacáridos/química , Dominios Proteicos , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.
Asunto(s)
Aminopiridinas/metabolismo , Benzodioxoles/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Indoles/metabolismo , Pliegue de Proteína , Aminopiridinas/química , Aminopiridinas/uso terapéutico , Animales , Benzodioxoles/química , Benzodioxoles/uso terapéutico , Sitios de Unión , Células CHO , Membrana Celular/química , Membrana Celular/metabolismo , Cricetulus , Microscopía por Crioelectrón , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/química , Indoles/uso terapéutico , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/uso terapéutico , Mutación , Dominios Proteicos/genética , Células Sf9 , TransfecciónRESUMEN
Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.
Asunto(s)
Mitocondrias/metabolismo , Degeneración Nerviosa/metabolismo , Mapas de Interacción de Proteínas , Sinapsis/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Aminoácidos/metabolismo , Biotinilación , Encéfalo/metabolismo , Encéfalo/patología , Núcleo Celular/metabolismo , Progresión de la Enfermedad , Metabolismo Energético , Demencia Frontotemporal/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Degeneración Nerviosa/patología , Neuronas/metabolismo , Unión Proteica , Dominios Proteicos , Proteómica , Índice de Severidad de la Enfermedad , Fracciones Subcelulares/metabolismo , Tauopatías/genética , Proteínas tau/químicaRESUMEN
The SARS-CoV-2 Omicron variant with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the spike (S) from Omicron reveals amino acid substitutions forging interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of the viral fusion step. Alterations in local conformation, charge, and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Structure of the Omicron S bound with human ACE2, together with the analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members, as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies, sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.
Asunto(s)
Evasión Inmune/fisiología , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Antivirales/inmunología , Sitios de Unión , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Microscopía por Crioelectrón , Humanos , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Unión Proteica , Dominios Proteicos/inmunología , Estructura Cuaternaria de Proteína , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Resonancia por Plasmón de Superficie , Acoplamiento ViralRESUMEN
Cullin-RING ubiquitin ligases (CRLs) are dynamic modular platforms that regulate myriad biological processes through target-specific ubiquitylation. Our knowledge of this system emerged from the F-box hypothesis, posited a quarter century ago: Numerous interchangeable F-box proteins confer specific substrate recognition for a core CUL1-based RING E3 ubiquitin ligase. This paradigm has been expanded through the evolution of a superfamily of analogous modular CRLs, with five major families and over 200 different substrate-binding receptors in humans. Regulation is achieved by numerous factors organized in circuits that dynamically control CRL activation and substrate ubiquitylation. CRLs also serve as a vast landscape for developing small molecules that reshape interactions and promote targeted ubiquitylation-dependent turnover of proteins of interest. Here, we review molecular principles underlying CRL function, the role of allosteric and conformational mechanisms in controlling substrate timing and ubiquitylation, and how the dynamics of substrate receptor interchange drives the turnover of selected target proteins to promote cellular decision-making.
Asunto(s)
Proteínas Cullin/química , Proteínas Cullin/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas F-Box/química , Retroalimentación Fisiológica , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteína NEDD8/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.
Asunto(s)
Proteínas SNARE/química , Proteínas SNARE/metabolismo , Enfermedad/genética , Humanos , Fusión de Membrana , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Mutación , Pinzas Ópticas , Fosforilación , Dominios Proteicos , Pliegue de Proteína , Proteínas SNARE/genéticaRESUMEN
Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.
Asunto(s)
Bioquímica/métodos , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Luz , Optogenética/métodos , Procesos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Fitocromo/química , Fitocromo/metabolismo , Dominios Proteicos , Ingeniería de Proteínas/métodos , Vitamina B 12/metabolismoRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Secuencia de Aminoácidos , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/ultraestructura , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
The NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here, we report that an endogenous, stimulus-responsive form of full-length mouse NLRP3 is a 12- to 16-mer double-ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for many NLRP3-activating stimuli, requires the double-ring cages of NLRP3. Double-ring-defective NLRP3 mutants abolish inflammasome punctum formation, caspase-1 processing, and cell death. Thus, our data uncover a physiological NLRP3 oligomer on the membrane that is poised to sense diverse signals to induce inflammasome activation.
Asunto(s)
Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Mutación/genética , Quinasas Relacionadas con NIMA/genética , Proteína con Dominio Pirina 3 de la Familia NLR/aislamiento & purificación , Proteína con Dominio Pirina 3 de la Familia NLR/ultraestructura , Nigericina/farmacología , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Red trans-Golgi/metabolismoRESUMEN
Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.
Asunto(s)
Arginina/análogos & derivados , Condensados Biomoleculares/metabolismo , Proteínas del Complejo SMN/química , Proteínas del Complejo SMN/metabolismo , Animales , Arginina/metabolismo , Núcleo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligandos , Metilación , Ratones , Modelos Biológicos , Células 3T3 NIH , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
The cholesterol-sensing protein Scap induces cholesterol synthesis by transporting membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi apparatus for proteolytic activation. Transport requires interaction between Scap's two ER luminal loops (L1 and L7), which flank an intramembrane sterol-sensing domain (SSD). Cholesterol inhibits Scap transport by binding to L1, which triggers Scap's binding to Insig, an ER retention protein. Here we used cryoelectron microscopy (cryo-EM) to elucidate two structures of full-length chicken Scap: (1) a wild-type free of Insigs and (2) mutant Scap bound to chicken Insig without cholesterol. Strikingly, L1 and L7 intertwine tightly to form a globular domain that acts as a luminal platform connecting the SSD to the rest of Scap. In the presence of Insig, this platform undergoes a large rotation accompanied by rearrangement of Scap's transmembrane helices. We postulate that this conformational change halts Scap transport of SREBPs and inhibits cholesterol synthesis.
Asunto(s)
Colesterol/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Pollos , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/ultraestructura , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , COVID-19/inmunología , Línea Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Unión Proteica/inmunología , Dominios Proteicos/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Células VeroRESUMEN
Selfish centromere DNA sequences bias their transmission to the egg in female meiosis. Evolutionary theory suggests that centromere proteins evolve to suppress costs of this "centromere drive." In hybrid mouse models with genetically different maternal and paternal centromeres, selfish centromere DNA exploits a kinetochore pathway to recruit microtubule-destabilizing proteins that act as drive effectors. We show that such functional differences are suppressed by a parallel pathway for effector recruitment by heterochromatin, which is similar between centromeres in this system. Disrupting the kinetochore pathway with a divergent allele of CENP-C reduces functional differences between centromeres, whereas disrupting heterochromatin by CENP-B deletion amplifies the differences. Molecular evolution analyses using Murinae genomes identify adaptive evolution in proteins in both pathways. We propose that centromere proteins have recurrently evolved to minimize the kinetochore pathway, which is exploited by selfish DNA, relative to the heterochromatin pathway that equalizes centromeres, while maintaining essential functions.
Asunto(s)
Proteína B del Centrómero/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Evolución Biológica , Sistemas CRISPR-Cas/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/química , Cromosomas de los Mamíferos/metabolismo , Femenino , Heterocromatina/metabolismo , Cinetocoros/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Oocitos/metabolismo , Dominios ProteicosRESUMEN
Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.
Asunto(s)
Fenómenos Electrofisiológicos , Transportadores de Sulfato/metabolismo , Aniones , Sitios de Unión , Cloruros/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Multimerización de Proteína , Estabilidad Proteica , Ácido Salicílico/metabolismo , Homología Estructural de Proteína , Transportadores de Sulfato/química , Transportadores de Sulfato/ultraestructuraRESUMEN
In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.