RESUMEN

Cryo-electron microscopy is a powerful methodology in structural biology and has been broadly used in high-resolution structure determination for challenging samples, which are not readily available for traditional techniques. In particular, the strength of super macro-complexes and the lack of a need for crystals for cryo-EM make this technique feasible for the structural study of complexes involved in antiviral innate immunity. This chapter presents detailed information and experimental procedures of Cryo-EM for determining the structures of the complexes using STING as an example. The procedures included a sample quality check, high-resolution data acquisition, and image processing for Cryo-EM 3D structure determination.

Asunto(s)

Microscopía por Crioelectrón , Inmunidad Innata , Microscopía por Crioelectrón/métodos , Humanos , Proteínas de la Membrana/inmunología , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos

RESUMEN

Fumonisin B1 (FB1) targets sphingolipid biosynthesis, inhibiting ceramide synthases. In this issue of Structure, Zhang et al.1 determined the cryoelectron microscopic structures of yeast ceramide synthase in complex with FB1 and its acylated derivative, acyl-FB1, revealing a two-step "ping-pong" mechanism for the N-acylation of FB1 and how it inhibits ceramide synthase.

Asunto(s)

Microscopía por Crioelectrón , Fumonisinas , Oxidorreductasas , Fumonisinas/química , Fumonisinas/metabolismo , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/antagonistas & inhibidores , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Acilación , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Esfingolípidos/metabolismo , Esfingolípidos/química

RESUMEN

Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. LEA proteins offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI-related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.

Asunto(s)

Aire , Microscopía por Crioelectrón , Congelación , Agua , Microscopía por Crioelectrón/métodos , Agua/química , Animales , Tomografía con Microscopio Electrónico/métodos , Caenorhabditis elegans

RESUMEN

Although aminergic GPCRs are the target for ~25% of approved drugs, developing subtype selective drugs is a major challenge due to the high sequence conservation at their orthosteric binding site. Bitopic ligands are covalently joined orthosteric and allosteric pharmacophores with the potential to boost receptor selectivity and improve current medications by reducing off-target side effects. However, the lack of structural information on their binding mode impedes rational design. Here we determine the cryo-EM structure of the hD3R:GαOßγ complex bound to the D3R selective bitopic agonist FOB02-04A. Structural, functional and computational analyses provide insights into its binding mode and point to a new TM2-ECL1-TM1 region, which requires the N-terminal ordering of TM1, as a major determinant of subtype selectivity in aminergic GPCRs. This region is underexploited in drug development, expands the established secondary binding pocket in aminergic GPCRs and could potentially be used to design novel and subtype selective drugs.

Asunto(s)

Microscopía por Crioelectrón , Receptores de Dopamina D3 , Humanos , Sitios de Unión , Receptores de Dopamina D3/metabolismo , Receptores de Dopamina D3/química , Receptores de Dopamina D3/agonistas , Células HEK293 , Ligandos , Unión Proteica , Animales , Modelos Moleculares

RESUMEN

Drug-resistant Tuberculosis (TB) is a global public health problem. Resistance to rifampicin, the most effective drug for TB treatment, is a major growing concern. The etiological agent, Mycobacterium tuberculosis (Mtb), has a cluster of ATP-binding cassette (ABC) transporters which are responsible for drug resistance through active export. Here, we describe studies characterizing Mtb Rv1217c-1218c as an ABC transporter that can mediate mycobacterial resistance to rifampicin and have determined the cryo-electron microscopy structures of Rv1217c-1218c. The structures show Rv1217c-1218c has a type V exporter fold. In the absence of ATP, Rv1217c-1218c forms a periplasmic gate by two juxtaposed-membrane helices from each transmembrane domain (TMD), while the nucleotide-binding domains (NBDs) form a partially closed dimer which is held together by four salt-bridges. Adenylyl-imidodiphosphate (AMPPNP) binding induces a structural change where the NBDs become further closed to each other, which downstream translates to a closed conformation for the TMDs. AMPPNP binding results in the collapse of the outer leaflet cavity and the opening of the periplasmic gate, which was proposed to play a role in substrate export. The rifampicin-bound structure shows a hydrophobic and periplasm-facing cavity is involved in rifampicin binding. Phospholipid molecules are observed in all determined structures and form an integral part of the Rv1217c-1218c transporter system. Our results provide a structural basis for a mycobacterial ABC exporter that mediates rifampicin resistance, which can lead to different insights into combating rifampicin resistance.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas , Microscopía por Crioelectrón , Farmacorresistencia Bacteriana , Mycobacterium tuberculosis , Rifampin , Rifampin/farmacología , Rifampin/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/ultraestructura , Transportadoras de Casetes de Unión a ATP/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Proteínas Bacterianas/genética , Modelos Moleculares , Adenilil Imidodifosfato/metabolismo

RESUMEN

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5' end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5' end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.

Asunto(s)

Microscopía por Crioelectrón , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/química , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , Conformación Proteica , Unión Proteica

RESUMEN

Protein filaments are ubiquitous in nature and have diverse biological functions. Cryo-electron microscopy (cryo-EM) enables the determination of atomic structures, even from native samples, and is capable of identifying previously unknown filament species through high-resolution cryo-EM maps. In this study, we determine the structure of an unreported filament species from a cryo-EM dataset collected from Bacillus amyloiquefaciens biofilms. These filaments are composed of GerQ, a spore coat protein known to be involved in Bacillus spore germination. GerQ assembles into a structurally stable architecture consisting of rings containing nine subunits, which stacks to form filaments. Molecular dockings and model predictions suggest that this nine-subunit structure is suitable for binding CwlJ, a protein recruited by GerQ and essential for Ca2+-DPA induced spore germination. While the assembly state of GerQ within the spores and the direct interaction between GerQ and CwlJ have yet to be validated through further experiments, our findings provide valuable insights into the self-assembly of GerQ and enhance our understanding of its role in spore germination.

Asunto(s)

Bacillus , Proteínas Bacterianas , Microscopía por Crioelectrón , Esporas Bacterianas , Microscopía por Crioelectrón/métodos , Esporas Bacterianas/ultraestructura , Esporas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Bacillus/metabolismo , Bacillus/genética , Simulación del Acoplamiento Molecular , Biopelículas/crecimiento & desarrollo , Unión Proteica

RESUMEN

Human vesicular monoamine transporter 2 (VMAT2), a member of the SLC18 family, plays a crucial role in regulating neurotransmitters in the brain by facilitating their uptake and storage within vesicles, preparing them for exocytotic release. Because of its central role in neurotransmitter signalling and neuroprotection, VMAT2 is a target for neurodegenerative diseases and movement disorders, with its inhibitor being used as therapeutics. Despite the importance of VMAT2 in pharmacophysiology, the molecular basis of VMAT2-mediated neurotransmitter transport and its inhibition remains unclear. Here we show the cryo-electron microscopy structure of VMAT2 in the substrate-free state, in complex with the neurotransmitter dopamine, and in complex with the inhibitor tetrabenazine. In addition to these structural determinations, monoamine uptake assays, mutational studies, and pKa value predictions were performed to characterize the dynamic changes in VMAT2 structure. These results provide a structural basis for understanding VMAT2-mediated vesicular transport of neurotransmitters and a platform for modulation of current inhibitor design.

Asunto(s)

Microscopía por Crioelectrón , Dopamina , Neurotransmisores , Tetrabenazina , Proteínas de Transporte Vesicular de Monoaminas , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/química , Humanos , Tetrabenazina/análogos & derivados , Tetrabenazina/metabolismo , Tetrabenazina/química , Dopamina/metabolismo , Neurotransmisores/metabolismo , Células HEK293 , Modelos Moleculares

RESUMEN

The yeast pre1-1(ß4-S142F) mutant accumulates late 20S proteasome core particle precursor complexes (late-PCs). We report a 2.1 Å cryo-EM structure of this intermediate with full-length Ump1 trapped inside, and Pba1-Pba2 attached to the α-ring surfaces. The structure discloses intimate interactions of Ump1 with ß2- and ß5-propeptides, which together fill most of the antechambers between the α- and ß-rings. The ß5-propeptide is unprocessed and separates Ump1 from ß6 and ß7. The ß2-propeptide is disconnected from the subunit by autocatalytic processing and localizes between Ump1 and ß3. A comparison of different proteasome maturation states reveals that maturation goes along with global conformational changes in the rings, initiated by structuring of the proteolytic sites and their autocatalytic activation. In the pre1-1 strain, ß2 is activated first enabling processing of ß1-, ß6-, and ß7-propeptides. Subsequent maturation of ß5 and ß1 precedes degradation of Ump1, tightening of the complex, and finally release of Pba1-Pba2.

Asunto(s)

Microscopía por Crioelectrón , Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Modelos Moleculares , Conformación Proteica , Péptidos/metabolismo , Péptidos/química , Unión Proteica , Chaperonas Moleculares

RESUMEN

Actin filament turnover involves subunits binding to and dissociating from the filament ends, with the pointed end being the primary site of filament disassembly. Several molecules modulate filament turnover, but the underlying mechanisms remain incompletely understood. Here, we present three cryo-EM structures of the F-actin pointed end in the presence and absence of phalloidin or DNase I. The two terminal subunits at the undecorated pointed end adopt a twisted conformation. Phalloidin can still bind and bridge these subunits, inducing a conformational shift to a flattened, F-actin-like state. This explains how phalloidin prevents depolymerization at the pointed end. Interestingly, two DNase I molecules simultaneously bind to the phalloidin-stabilized pointed end. In the absence of phalloidin, DNase I binding would disrupt the terminal actin subunit packing, resulting in filament disassembly. Our findings uncover molecular principles of pointed end regulation and provide structural insights into the kinetic asymmetry between the actin filament ends.

Asunto(s)

Citoesqueleto de Actina , Actinas , Microscopía por Crioelectrón , Desoxirribonucleasa I , Faloidina , Actinas/metabolismo , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasa I/química , Citoesqueleto de Actina/metabolismo , Faloidina/metabolismo , Faloidina/química , Modelos Moleculares , Unión Proteica , Animales , Conejos , Conformación Proteica

RESUMEN

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography - a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) - has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we apply 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employ low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allows us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples are iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 Å resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions.

Asunto(s)

Microscopía por Crioelectrón , Relación Señal-Ruido , Microscopía por Crioelectrón/métodos , Microscopía Electrónica de Transmisión de Rastreo/métodos , Imagenología Tridimensional/métodos , Apoferritinas/química , Apoferritinas/ultraestructura , Proteínas/química , Proteínas/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos

RESUMEN

Heart failure, the leading cause of mortality and morbidity in the developed world, is characterized by cardiac ryanodine receptor 2 channels that are hyperphosphorylated, oxidized, and depleted of the stabilizing subunit calstabin-2. This results in a diastolic sarcoplasmic reticulum Ca2+ leak that impairs cardiac contractility and triggers arrhythmias. Genetic mutations in ryanodine receptor 2 can also cause Ca2+ leak, leading to arrhythmias and sudden cardiac death. Here, we solved the cryogenic electron microscopy structures of ryanodine receptor 2 variants linked either to heart failure or inherited sudden cardiac death. All are in the primed state, part way between closed and open. Binding of Rycal drugs to ryanodine receptor 2 channels reverts the primed state back towards the closed state, decreasing Ca2+ leak, improving cardiac function, and preventing arrhythmias. We propose a structural-physiological mechanism whereby the ryanodine receptor 2 channel primed state underlies the arrhythmias in heart failure and arrhythmogenic disorders.

Asunto(s)

Arritmias Cardíacas , Calcio , Microscopía por Crioelectrón , Insuficiencia Cardíaca , Canal Liberador de Calcio Receptor de Rianodina , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/química , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/genética , Humanos , Animales , Calcio/metabolismo , Mutación , Retículo Sarcoplasmático/metabolismo , Muerte Súbita Cardíaca/etiología

RESUMEN

The filamentous fungus Magnaporthe oryzae is widely recognized as a notorious plant pathogen responsible for causing rice blasts. With rapid advancements in molecular biology technologies, numerous regulatory mechanisms have been thoroughly investigated. However, most recent studies have predominantly focused on infection-related pathways or host defence mechanisms, which may be insufficient for developing novel structure-based prevention strategies. A substantial body of literature has utilized cryo-electron microscopy and X-ray diffraction to explore the relationships between functional components, shedding light on the identification of potential drug targets. Owing to the complexity of protein extraction and stochastic nature of crystallization, obtaining high-quality structures remains a significant challenge for the scientific community. Emerging computational tools such as AlphaFold for structural prediction, docking for interaction analysis, and molecular dynamics simulations to replicate in vivo conditions provide novel avenues for overcoming these challenges. In this review, we aim to consolidate the structural biological advancements in M. oryzae, drawing upon mature experimental experiences from other species such as Saccharomyces cerevisiae and mammals. We aim to explore the potential of protein construction to address the invasion and proliferation of M. oryzae, with the goal of identifying new drug targets and designing small-molecule compounds to manage this disease.

Asunto(s)

Proteínas Fúngicas , Oryza , Enfermedades de las Plantas , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ascomicetos/genética , Ascomicetos/patogenicidad , Ascomicetos/química , Microscopía por Crioelectrón

RESUMEN

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded ß-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Pliegue de Proteína , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/genética , Microscopía por Crioelectrón , Unión Proteica , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/química , Mutación , Proteínas Portadoras , Isomerasa de Peptidilprolil

RESUMEN

WNT signaling is fundamental in development and homeostasis, but how the Frizzled receptors (FZDs) propagate signaling remains enigmatic. Here, we present the cryo-EM structure of FZD4 engaged with the DEP domain of Dishevelled 2 (DVL2), a key WNT transducer. We uncover a distinct binding mode where the DEP finger-loop inserts into the FZD4 cavity to form a hydrophobic interface. FZD4 intracellular loop 2 (ICL2) additionally anchors the complex through polar contacts. Mutagenesis validates the structural observations. The DEP interface is highly conserved in FZDs, indicating a universal mechanism by which FZDs engage with DVLs. We further reveal that DEP mimics G-protein/ß-arrestin/GRK to recognize an active conformation of receptor, expanding current GPCR engagement models. Finally, we identify a distinct FZD4 dimerization interface. Our findings delineate the molecular determinants governing FZD/DVL assembly and propagation of WNT signaling, providing long-sought answers underlying WNT signal transduction.

Asunto(s)

Proteínas Dishevelled , Receptores Frizzled , Vía de Señalización Wnt , Receptores Frizzled/metabolismo , Receptores Frizzled/química , Receptores Frizzled/genética , Proteínas Dishevelled/metabolismo , Proteínas Dishevelled/genética , Proteínas Dishevelled/química , Humanos , Células HEK293 , Unión Proteica , Microscopía por Crioelectrón , Modelos Moleculares , Dominios Proteicos

RESUMEN

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.

Asunto(s)

Citomegalovirus , Proteínas del Envoltorio Viral , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Citomegalovirus/inmunología , Humanos , Animales , Ratones , Microscopía por Crioelectrón , Conformación Proteica , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Internalización del Virus , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Estabilidad Proteica , Vacunas contra Citomegalovirus/inmunología , Sustitución de Aminoácidos , Modelos Moleculares

RESUMEN

The fusion of viruses with cellular membranes is a critical step in the life cycle of enveloped viruses. This process is facilitated by viral fusion proteins, many of which are conformationally pH-sensitive. The specifics of how changes in pH initiate this fusion have remained largely elusive. This study presents the cryo-electron microscopy (cryo-EM) structures of a prototype class III fusion protein, GP64, in its prefusion and early intermediate states, revealing the structural intermediates accompanying the membrane fusion process. The structures identify the involvement of a pH-sensitive switch, comprising H23, H245, and H304, in sensing the low pH that triggers the initial step of membrane fusion. The pH sensing role of this switch is corroborated by assays of cell-cell syncytium formation and dual dye-labeling. The findings demonstrate that coordination between multiple histidine residues acts as a pH sensor and activator. The involvement of a multi-histidine switch in viral fusion is applicable to fusogens of human-infecting thogotoviruses and other viruses, which could lead to strategies for developing anti-viral therapies and vaccines.

Asunto(s)

Microscopía por Crioelectrón , Histidina , Concentración de Iones de Hidrógeno , Histidina/química , Histidina/metabolismo , Humanos , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo , Fusión de Membrana , Animales , Modelos Moleculares , Internalización del Virus , Conformación Proteica

RESUMEN

Nascent chains undergo co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common combination of subsequent modifications carried out on the 80S ribosome. How these enzymatic processes are coordinated in the context of a rapidly translating ribosome has remained elusive. Here, we report two cryo-EM structures of multi-enzyme complexes assembled on vacant human 80S ribosomes, indicating two routes for NME-NTA. Both assemblies form on the 80S independent of nascent chain substrates. Irrespective of the route, NatA occupies a non-intrusive 'distal' binding site on the ribosome which does not interfere with MAP1 or MAP2 binding nor with most other ribosome-associated factors (RAFs). NatA can partake in a coordinated, dynamic assembly with MAP1 through the hydra-like chaperoning function of the abundant Nascent Polypeptide-Associated Complex (NAC). In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Together, our data provide the structural framework for the coordinated orchestration of NME and NTA in protein biogenesis.

Asunto(s)

Microscopía por Crioelectrón , Ribosomas , Humanos , Ribosomas/metabolismo , Acetilación , Procesamiento Proteico-Postraduccional , Sitios de Unión , Biosíntesis de Proteínas , Unión Proteica , Metionina/metabolismo , Modelos Moleculares

RESUMEN

The Omicron subvariants of SARS-CoV-2, especially for BA.2.86 and JN.1, have rapidly spread across multiple countries, posing a significant threat in the ongoing COVID-19 pandemic. Distinguished by 34 additional mutations on the Spike (S) protein compared to its BA.2 predecessor, the implications of BA.2.86 and its evolved descendant, JN.1 with additional L455S mutation in receptor-binding domains (RBDs), are of paramount concern. In this work, we systematically examine the neutralization susceptibilities of SARS-CoV-2 Omicron subvariants and reveal the enhanced antibody evasion of BA.2.86 and JN.1. We also determine the cryo-EM structures of the trimeric S proteins from BA.2.86 and JN.1 in complex with the host receptor ACE2, respectively. The mutations within the RBDs of BA.2.86 and JN.1 induce a remodeling of the interaction network between the RBD and ACE2. The L455S mutation of JN.1 further induces a notable shift of the RBD-ACE2 interface, suggesting the notably reduced binding affinity of JN.1 than BA.2.86. An analysis of the broadly neutralizing antibodies possessing core neutralizing epitopes reveals the antibody evasion mechanism underlying the evolution of Omicron BA.2.86 subvariant. In general, we construct a landscape of evolution in virus-receptor of the circulating Omicron subvariants.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Microscopía por Crioelectrón , Evasión Inmune , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Humanos , COVID-19/inmunología , COVID-19/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/química , Evolución Molecular , Unión Proteica , Modelos Moleculares

RESUMEN

The tropomyosin 1 isoform I/C C-terminal domain (Tm1-LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo-EM) and solid state nuclear magnetic resonance (NMR). This study demonstrates the complementary nature of these two structural biology techniques. Chemical shift assignments from solid state NMR are used to determine the secondary structure at the level of individual amino acids, which is faithfully seen in cryo-EM reconstructions. Additionally, solid state NMR demonstrates that the region not observed in the reconstructed cryo-EM density is primarily in a highly mobile random coil conformation rather than adopting multiple rigid conformations. Overall, this study illustrates the benefit of investigations combining cryo-EM and solid state NMR to investigate protein fibril structure.

Asunto(s)

Microscopía por Crioelectrón , Resonancia Magnética Nuclear Biomolecular , Tropomiosina , Microscopía por Crioelectrón/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Tropomiosina/química , Tropomiosina/ultraestructura , Modelos Moleculares , Estructura Secundaria de Proteína , Conformación Proteica