RESUMO
During the last ten years, developments in cryo-electron microscopy have transformed our understanding of eukaryotic ribosome assembly. As a result, the field has advanced from a list of the vast array of ribosome assembly factors toward an emerging molecular movie in which individual frames are represented by structures of stable ribosome assembly intermediates with complementary biochemical and genetic data. In this review, we discuss the mechanisms driving the assembly of yeast and human small and large ribosomal subunits. A particular emphasis is placed on the most recent findings that illustrate key concepts of ribosome assembly, such as folding of preribosomal RNA, the enforced chronology of assembly, enzyme-mediated irreversible transitions, and proofreading of preribosomal particles.
Assuntos
Microscopia Crioeletrônica , Proteínas Ribossômicas , Ribossomos , Humanos , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Ribossomos/química , Ribossomos/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Modelos Moleculares , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Dobramento de RNA , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , AnimaisRESUMO
Positive-strand RNA viruses encompass a variety of established and emerging eukaryotic pathogens. Their genome replication is confined to specialized cytoplasmic membrane compartments known as replication organelles (ROs). These ROs derive from host membranes, transformed into distinct structures such as invaginated spherules or intricate membrane networks including single- and/or double-membrane vesicles. ROs play a vital role in orchestrating viral RNA synthesis and evading detection by innate immune sensors of the host. In recent years, groundbreaking cryo-electron microscopy studies conducted with several prototypic viruses have significantly advanced our understanding of RO structure and function. Notably, these studies unveiled the presence of crown-shaped multimeric viral protein complexes that seem to actively participate in viral RNA synthesis and regulate the release of newly synthesized RNA into the cytosol for translation and packaging. These findings have shed light on novel viral functions and fascinating macromolecular complexes that delineate promising new avenues for future research.
Assuntos
Microscopia Crioeletrônica , RNA Viral , Replicação Viral , Microscopia Crioeletrônica/métodos , RNA Viral/metabolismo , RNA Viral/genética , RNA Viral/química , Humanos , Vírus de RNA de Cadeia Positiva/metabolismo , Vírus de RNA de Cadeia Positiva/genética , Vírus de RNA de Cadeia Positiva/química , Vírus de RNA de Cadeia Positiva/ultraestrutura , Organelas/ultraestrutura , Organelas/virologia , Organelas/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/ultraestrutura , Animais , Compartimentos de Replicação Viral/metabolismo , Compartimentos de Replicação Viral/ultraestruturaRESUMO
Mastigonemes, the hair-like lateral appendages lining cilia or flagella, participate in mechanosensation and cellular motion, but their constituents and structure have remained unclear. Here, we report the cryo-EM structure of native mastigonemes isolated from Chlamydomonas at 3.0 Å resolution. The long stem assembles as a super spiral, with each helical turn comprising four pairs of anti-parallel mastigoneme-like protein 1 (Mst1). A large array of arabinoglycans, which represents a common class of glycosylation in plants and algae, is resolved surrounding the type II poly-hydroxyproline (Hyp) helix in Mst1. The EM map unveils a mastigoneme axial protein (Mstax) that is rich in heavily glycosylated Hyp and contains a PKD2-like transmembrane domain (TMD). Mstax, with nearly 8,000 residues spanning from the intracellular region to the distal end of the mastigoneme, provides the framework for Mst1 assembly. Our study provides insights into the complexity of protein and glycan interactions in native bio-architectures.
Assuntos
Chlamydomonas , Cílios , Chlamydomonas/citologia , Cílios/química , Cílios/ultraestrutura , Flagelos , Polissacarídeos , ProteínasRESUMO
High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly.
Assuntos
Drosophila melanogaster , Microscopia Eletrônica , Neurotransmissores , Sinapses , Animais , Encéfalo/ultraestrutura , Encéfalo/metabolismo , Conectoma , Drosophila melanogaster/ultraestrutura , Drosophila melanogaster/metabolismo , Ácido gama-Aminobutírico/metabolismo , Microscopia Eletrônica/métodos , Redes Neurais de Computação , Neurônios/metabolismo , Neurônios/ultraestrutura , Neurotransmissores/metabolismo , Sinapses/ultraestrutura , Sinapses/metabolismoRESUMO
The very-low-density lipoprotein receptor (VLDLR) comprises eight LDLR type A (LA) domains and supports entry of distantly related alphaviruses, including Eastern equine encephalitis virus (EEEV) and Semliki Forest virus (SFV). Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage more than one LA domain simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection. Whereas all EEEV strains show conservation of two VLDLR-binding sites, the EEEV PE-6 strain and a few other EEE complex members feature a single amino acid substitution that enables binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.
Assuntos
Microscopia Crioeletrônica , Vírus da Encefalite Equina do Leste , Encefalomielite Equina , Receptores de LDL , Animais , Camundongos , Alphavirus/fisiologia , Vírus da Encefalite Equina do Leste/fisiologia , Vírus da Encefalite Equina do Leste/ultraestrutura , Encefalomielite Equina/metabolismo , Cavalos , Ligação Proteica , Receptores de LDL/ultraestruturaRESUMO
Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.
Assuntos
Gametogênese , Mitocôndrias , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Coenzima A Ligases/metabolismo , Microscopia Crioeletrônica , Citoplasma/metabolismo , Tomografia com Microscopia Eletrônica , Meiose , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Esporos Fúngicos/metabolismo , Modelos Moleculares , Estrutura Quaternária de ProteínaRESUMO
The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.
Assuntos
Receptor Nicotínico de Acetilcolina alfa7 , Humanos , Receptor Nicotínico de Acetilcolina alfa7/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Inflamação/tratamento farmacológico , Transdução de Sinais , Regulação AlostéricaRESUMO
Bidirectional transport in cilia is carried out by polymers of the IFTA and IFTB protein complexes, called anterograde and retrograde intraflagellar transport (IFT) trains. Anterograde trains deliver cargoes from the cell to the cilium tip, then convert into retrograde trains for cargo export. We set out to understand how the IFT complexes can perform these two directly opposing roles before and after conversion. We use cryoelectron tomography and in situ cross-linking mass spectrometry to determine the structure of retrograde IFT trains and compare it with the known structure of anterograde trains. The retrograde train is a 2-fold symmetric polymer organized around a central thread of IFTA complexes. We conclude that anterograde-to-retrograde remodeling involves global rearrangements of the IFTA/B complexes and requires complete disassembly of the anterograde train. Finally, we describe how conformational changes to cargo-binding sites facilitate unidirectional cargo transport in a bidirectional system.
Assuntos
Cílios , Microscopia Crioeletrônica , Flagelos , Flagelos/metabolismo , Flagelos/ultraestrutura , Cílios/metabolismo , Transporte Biológico , Chlamydomonas reinhardtii/metabolismo , Modelos Moleculares , Transporte ProteicoRESUMO
Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.
Assuntos
Vírus Bluetongue , Proteínas do Capsídeo , Capsídeo , Microscopia Crioeletrônica , RNA Viral , Empacotamento do Genoma Viral , Vírus Bluetongue/genética , Vírus Bluetongue/fisiologia , Vírus Bluetongue/metabolismo , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Animais , RNA Viral/metabolismo , RNA Viral/genética , Genoma Viral/genética , Montagem de Vírus , Tomografia com Microscopia Eletrônica , Vírion/metabolismo , Vírion/genética , Vírion/ultraestrutura , Modelos Moleculares , Linhagem Celular , CricetinaeRESUMO
The glycine transporter 1 (GlyT1) plays a crucial role in the regulation of both inhibitory and excitatory neurotransmission by removing glycine from the synaptic cleft. Given its close association with glutamate/glycine co-activated NMDA receptors (NMDARs), GlyT1 has emerged as a central target for the treatment of schizophrenia, which is often linked to hypofunctional NMDARs. Here, we report the cryo-EM structures of GlyT1 bound with substrate glycine and drugs ALX-5407, SSR504734, and PF-03463275. These structures, captured at three fundamental states of the transport cycle-outward-facing, occluded, and inward-facing-enable us to illustrate a comprehensive blueprint of the conformational change associated with glycine reuptake. Additionally, we identified three specific pockets accommodating drugs, providing clear insights into the structural basis of their inhibitory mechanism and selectivity. Collectively, these structures offer significant insights into the transport mechanism and recognition of substrate and anti-schizophrenia drugs, thus providing a platform to design small molecules to treat schizophrenia.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina , Humanos , Transporte Biológico , Glicina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/química , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/ultraestrutura , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/metabolismo , Transmissão Sináptica , Imidazóis/química , Sarcosina/análogos & derivados , Piperidinas/químicaRESUMO
Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.
Assuntos
Receptores de Apelina , Fármacos Cardiovasculares , Desenho de Fármacos , Receptores de Apelina/agonistas , Receptores de Apelina/química , Receptores de Apelina/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Humanos , Fármacos Cardiovasculares/químicaRESUMO
Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.
Assuntos
Microscopia Crioeletrônica , Spumavirus , Montagem de Vírus , Internalização do Vírus , Spumavirus/genética , Capsídeo/metabolismo , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Humanos , Evolução Molecular , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Modelos MolecularesRESUMO
Semliki Forest virus (SFV) is an alphavirus that uses the very-low-density lipoprotein receptor (VLDLR) as a receptor during infection of its vertebrate hosts and insect vectors. Herein, we used cryoelectron microscopy to study the structure of SFV in complex with VLDLR. We found that VLDLR binds multiple E1-DIII sites of SFV through its membrane-distal LDLR class A (LA) repeats. Among the LA repeats of the VLDLR, LA3 has the best binding affinity to SFV. The high-resolution structure shows that LA3 binds SFV E1-DIII through a small surface area of 378 Å2, with the main interactions at the interface involving salt bridges. Compared with the binding of single LA3s, consecutive LA repeats around LA3 promote synergistic binding to SFV, during which the LAs undergo a rotation, allowing simultaneous key interactions at multiple E1-DIII sites on the virion and enabling the binding of VLDLRs from divergent host species to SFV.
Assuntos
Receptores de LDL , Vírus da Floresta de Semliki , Alphavirus/metabolismo , Microscopia Crioeletrônica , Vírus da Floresta de Semliki/metabolismo , Vírus da Floresta de Semliki/ultraestrutura , Receptores de LDL/metabolismo , Receptores de LDL/ultraestrutura , Receptores Virais/metabolismo , Receptores Virais/ultraestruturaRESUMO
Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.
Assuntos
Ascomicetos , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , Técnicas de Introdução de Genes , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/ultraestrutura , Microscopia Crioeletrônica , Ascomicetos/química , Ascomicetos/metabolismo , Ascomicetos/ultraestruturaRESUMO
KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.
Assuntos
Channelrhodopsins , Rhinosporidium , Humanos , Channelrhodopsins/química , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Channelrhodopsins/ultraestrutura , Microscopia Crioeletrônica , Canais Iônicos , Potássio/metabolismo , Rhinosporidium/químicaRESUMO
The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.
Assuntos
Proteína ADAM10 , Animais , Proteína ADAM10/química , Proteína ADAM10/metabolismo , Proteína ADAM10/ultraestrutura , Secretases da Proteína Precursora do Amiloide/química , Mamíferos/metabolismo , Proteólise , Tetraspaninas/metabolismo , HumanosRESUMO
The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.
Assuntos
Ibogaína , Inibidores Seletivos de Recaptação de Serotonina , Proteínas da Membrana Plasmática de Transporte de Serotonina , Bibliotecas de Moléculas Pequenas , Animais , Camundongos , Fluoxetina/farmacologia , Ibogaína/química , Ibogaína/farmacologia , Conformação Molecular , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/ultraestrutura , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Vírion/ultraestrutura , Produtos do Gene env do Vírus da Imunodeficiência Humana/ultraestrutura , Produtos do Gene gag do Vírus da Imunodeficiência Humana/ultraestrutura , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacologia , Sequência de Aminoácidos , Dissulfetos/farmacologia , Epitopos/química , Células HEK293 , Proteína gp41 do Envelope de HIV/química , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Modelos Moleculares , Testes de Neutralização , Peptídeos/química , Polissacarídeos/química , Domínios Proteicos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/químicaRESUMO
Transmembrane ß barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the ß barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model ß barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "ß signal" motif of EspP to correctly orient ß strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated ß sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that ß sheets progressively fold toward BamA to form a ß barrel. Along with in vivo experiments that tracked ß barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate ß barrel folding.
Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/metabolismo , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.