RESUMEN
T cells in jawed vertebrates comprise two lineages, αß T-cells and γδ T-cells, defined by the antigen receptors they express, i.e., αß and γδ T-cell receptors (TCRs), respectively. The two lineages have different immunological roles, requiring γδ TCRs to recognize more structurally-diverse ligands1. Nevertheless, the receptors use shared CD3 subunits to initiate signaling. Whereas the structural organization of αß TCRs is understood2,3, the architecture of γδ TCRs is unknown. Here, we used cryogenic electron microscopy to determine the structure of a fully-assembled, MR1-reactive human Vδ3Vγ8 TCR/CD3δγε2ζ2 complex bound by anti-CD3ε antibody Fab fragments4,5. The arrangement of CD3 subunits in γδ and αß TCRs is conserved and, although the transmembrane α-helices of the TCR-γδ and -αß subunits differ markedly in sequence, the packing of the eight transmembrane-helix bundles is similar6. However, in contrast to the apparently rigid αß TCR2,3,6, the γδ TCR exhibits considerable conformational heterogeneity, owing to the ligand-binding TCR-γδ subunits being tethered to the CD3 subunits by their transmembrane regions only. Reducing this conformational heterogeneity by transferring the Vδ3Vγ8 TCR variable domains to an αß TCR enhanced receptor signaling, suggesting that γδ TCR organization reflects a compromise between efficient signaling and the ability to engage structurally-diverse ligands. Our findings reveal the remarkable structural plasticity of the TCR on evolutionary timescales, and recast it as a highly versatile receptor capable of initiating signaling as either a rigid or flexible structure.
RESUMEN
Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.
Asunto(s)
Antibacterianos/biosíntesis , Furanos/metabolismo , Streptomyces coelicolor/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN/metabolismo , ADN/ultraestructura , Furanos/química , Hormonas/química , Hormonas/clasificación , Hormonas/metabolismo , Ligandos , Modelos Moleculares , Péptidos/metabolismo , Proteínas Represoras/química , Proteínas Represoras/clasificación , Proteínas Represoras/metabolismo , Proteínas Represoras/ultraestructura , Transducción de Señal , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Relación Estructura-ActividadRESUMEN
The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.
Asunto(s)
Analgesia , Receptor de Adenosina A1/metabolismo , Adenosina/química , Adenosina/metabolismo , Regulación Alostérica/efectos de los fármacos , Analgesia/métodos , Animales , Sitios de Unión , Modelos Animales de Enfermedad , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2/química , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Hiperalgesia/tratamiento farmacológico , Lípidos , Masculino , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Estabilidad Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A1/química , Transducción de Señal/efectos de los fármacosRESUMEN
Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.
Asunto(s)
Activación Plaquetaria , Trombocitopenia , Estados Unidos , Humanos , Microscopía por Crioelectrón , Ácido Araquidónico/metabolismo , Araquidonato 12-Lipooxigenasa/metabolismoRESUMEN
The class A orphan G protein-coupled receptor (GPCR), GPR3, has been implicated in a variety of conditions, including Alzheimer's and premature ovarian failure. GPR3 constitutively couples with Gαs, resulting in the production of cAMP in cells. While tool compounds and several putative endogenous ligands have emerged for the receptor, its endogenous ligand, if it exists, remains a mystery. As novel potential drug targets, the structures of orphan GPCRs have been of increasing interest, revealing distinct modes of activation, including autoactivation, presence of constitutively activating mutations, or via cryptic ligands. Here, we present a cryo-electron microscopy (cryo-EM) structure of the orphan GPCR, GPR3 in complex with DNGαs and Gß1γ2. The structure revealed clear density for a lipid-like ligand that bound within an extended hydrophobic groove, suggesting that the observed "constitutive activity" was likely due to activation via a lipid that may be ubiquitously present. Analysis of conformational variance within the cryo-EM data set revealed twisting motions of the GPR3 transmembrane helices that appeared coordinated with changes in the lipid-like density. We propose a mechanism for the binding of a lipid to its putative orthosteric binding pocket linked to the GPR3 dynamics.
Asunto(s)
Lípidos , Receptores Acoplados a Proteínas G , Ligandos , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismoRESUMEN
The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound ß2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides molecular insights into receptor and G-protein selectivity.
Asunto(s)
Adenosina/química , Adenosina/metabolismo , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Receptor de Adenosina A1/química , Receptor de Adenosina A1/ultraestructura , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Modelos Moleculares , Receptor de Adenosina A1/metabolismo , Rotación , Especificidad por SustratoRESUMEN
Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Tratamiento Farmacológico de COVID-19 , COVID-19 , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , COVID-19/inmunología , Camélidos del Nuevo Mundo , Humanos , Ratones , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacologíaRESUMEN
M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and Plasmodium vivax (Pv-M17) function as catalytically active hexamers to generate free amino acids from human hemoglobin and are drug targets for the design of novel antimalarial agents. However, the molecular basis for oligomeric assembly is not fully understood. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found that PfA-M17 and Pv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species that can be controlled by manipulating the identity and concentration of metals available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of Pv-M17 by cryoelectron microscopy and X-ray crystallography together with solution studies revealed that PfA-M17 and Pv-M17 bind metal ions and substrates in a conserved fashion, although Pv-M17 forms the active hexamer more readily and processes substrates faster than PfA-M17. On the basis of these studies, we propose a dynamic equilibrium between monomer â dimer â tetramer â hexamer, which becomes directional toward the large oligomeric states with the addition of metal ions. This sophisticated metal-dependent dynamic equilibrium may apply to other M17 aminopeptidases and underpin the moonlighting capabilities of this enzyme family.
Asunto(s)
Aminopeptidasas/química , Manganeso/química , Plasmodium falciparum/enzimología , Plasmodium vivax/enzimología , Multimerización de Proteína , Proteínas Protozoarias/química , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Dominio Catalítico , Cationes Bivalentes , Clonación Molecular , Cobalto/química , Cobalto/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Dipéptidos/química , Dipéptidos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Magnesio/química , Magnesio/metabolismo , Manganeso/metabolismo , Modelos Moleculares , Mutación , Plasmodium falciparum/genética , Plasmodium vivax/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Zinc/química , Zinc/metabolismoRESUMEN
DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine-guanosine oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here, we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH-dependent oligomerization pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerization pathway within a cellular setting, whereby cytosine-guanosine binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.
Asunto(s)
Antígenos CD/química , Antígenos CD/metabolismo , Microscopía por Crioelectrón/métodos , Fibronectinas/metabolismo , Lectinas Tipo C/metabolismo , Antígenos de Histocompatibilidad Menor/química , Antígenos de Histocompatibilidad Menor/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Humanos , Lectinas Tipo C/química , Ligandos , Conformación ProteicaRESUMEN
The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.
Asunto(s)
Iniciación de la Cadena Peptídica Traduccional , Poliovirus/genética , ARN Viral/química , Proteínas de Unión al ARN/química , Microscopía por Crioelectrón , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
We developed a series of highly crystalline double-cable conjugated polymers for application in single-component organic solar cells (SCOSCs). These polymers contain conjugated backbones as electron donor and pendant perylene bisimide units (PBIs) as electron acceptor. PBIs are connected to the backbone via alkyl units varying from hexyl (C6 H12 ) to eicosyl (C20 H40 ) as flexible linkers. For double-cable polymers with short linkers, the PBIs tend to stack in a head-to-head fashion, resulting in large d-spacings (e.g. 64â Å for the polymer P12 with C12 H24 linker) along the lamellar stacking direction. When the length of the linker groups is longer than a certain length, the PBIs instead adopt a more ordered packing likely via H-aggregation, resulting in short d-spacings (e.g. 50â Å for the polymer P16 with C16 H32 linker). This work highlights the importance of linker length on the molecular packing of the acceptor units and the influences on the photovoltaic performance of SCOSCs.
RESUMEN
Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.
Asunto(s)
Microscopía por Crioelectrón/métodos , Microscopía Electrónica de Transmisión/métodos , Animales , Tomografía con Microscopio Electrónico/métodos , HumanosRESUMEN
The efficiency of an organic solar cell is highly dependent on the complex, interpenetrating morphology, and molecular order within the composite phases of the bulk heterojunction (BHJ) blend. Both these microstructural aspects are strongly influenced by the processing conditions and chemical design of donor/acceptor materials. To establish improved structure-function relationships, it is vital to visualize the local microstructural order to provide specific local information about donor/acceptor interfaces and crystalline texture in BHJ blend films. The visualization of nanocrystallites, however, is difficult due to the complex semi-crystalline structure with few characterization techniques capable of visualizing the molecular ordering of soft materials at the nanoscale. Here, it is demonstrated how cryo-electron microscopy can be utilized to visualize local nanoscale order. This method is used to understand the distribution/orientation of crystallites in a BHJ blend. Long-range (>300 nm) texturing of IEICO-4F crystallites oriented in an edge-on fashion is observed, which has not previously been observed for spin-coated materials. This approach provides a wealth of quantitative information about the texture and size of nanocrystallites, which can be utilized to understand charge generation and transport in organic film. This study guides tailoring the material design and processing conditions for high-performance organic optoelectronic devices.
RESUMEN
Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.
RESUMEN
The Membrane Attack Complex (MAC) is responsible for forming large ß-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.
Asunto(s)
Complemento C9 , Complejo de Ataque a Membrana del Sistema Complemento , Humanos , Animales , Ratones , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Complemento C5b , Complemento C9/química , Complemento C9/metabolismo , Proteínas del Sistema Complemento/metabolismo , EpítoposRESUMEN
The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin ß, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin ß reveal unique features of the active site of meprin α, and helical assembly more broadly.
Asunto(s)
Fetuína-B , Metaloendopeptidasas , Humanos , Microscopía por Crioelectrón , Metaloendopeptidasas/metabolismo , Metaloproteasas , Precursores Enzimáticos , ZincRESUMEN
Bacteriocins are bacterial peptides with specific activity against competing species. They hold great potential as natural preservatives and for their probiotic effects. We show here nuclear magnetic resonance-based evidence that glycocin F, a 43-amino acid bacteriocin from Lactobacillus plantarum, contains two ß-linked N-acetylglucosamine moieties, attached via side chain linkages to a serine via oxygen, and to a cysteine via sulfur. The latter linkage is novel and has helped to establish a new type of post-translational modification, the S-linked sugar. The peptide conformation consists primarily of two α-helices held together by a pair of nested disulfide bonds. The serine-linked sugar is positioned on a short loop sequentially connecting the two helices, while the cysteine-linked sugar presents at the end of a long disordered C-terminal tail. The differing chemical and conformational stabilities of the two N-actetylglucosamine moieties provide clues about the possible mode of action of this bacteriostatic peptide.
Asunto(s)
Bacteriocinas/química , Espectroscopía de Resonancia Magnética/métodos , Conformación Proteica , Estructura Secundaria de Proteína , Acetilglucosamina/química , Bacteriocinas/metabolismo , Cisteína/química , Disulfuros/química , Glicosilación , Cinética , Lactobacillus plantarum/metabolismo , Modelos Moleculares , Oxígeno/química , Procesamiento Proteico-Postraduccional , Serina/química , Azufre/químicaRESUMEN
Flaviviruses are the cause of severe human diseases transmitted by mosquitoes and ticks. These viruses use a potent fusion machinery to enter target cells that needs to be restrained during viral assembly and egress. A molecular chaperone, premembrane (prM) maintains the virus particles in an immature, fusion-incompetent state until they exit the cell. Taking advantage of an insect virus that produces particles that are both immature and infectious, we determined the structure of the first immature flavivirus with a complete spike by cryo-electron microscopy. Unexpectedly, the prM chaperone forms a supporting pillar that maintains the immature spike in an asymmetric and upright state, primed for large rearrangements upon acidification. The collapse of the spike along a path defined by the prM chaperone is required, and its inhibition by a multivalent immunoglobulin M blocks infection. The revised architecture and collapse model are likely to be conserved across flaviviruses.
RESUMEN
The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.
Asunto(s)
Infecciones por Flavivirus/terapia , Flavivirus/ultraestructura , Proteínas del Envoltorio Viral/ultraestructura , Virión/ultraestructura , Aedes/virología , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Chlorocebus aethiops , Microscopía por Crioelectrón , Dengue/terapia , Dengue/virología , Vacunas contra el Dengue/administración & dosificación , Vacunas contra el Dengue/farmacología , Diseño de Fármacos , Flavivirus/efectos de los fármacos , Flavivirus/inmunología , Flavivirus/patogenicidad , Infecciones por Flavivirus/virología , Humanos , Mesocricetus , Modelos Moleculares , Conformación Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/farmacología , Vacunas Virales/uso terapéutico , Virión/efectos de los fármacos , Virión/metabolismoRESUMEN
Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.