RESUMEN
The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively. In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit. The structure and accompanying factor-binding data show that CrPV-IRES binding mimics a pretranslocation rather than initiation state of the ribosome. Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.
Asunto(s)
Dicistroviridae/química , Kluyveromyces/química , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/química , ARN Viral/química , Ribosomas/química , Microscopía por Crioelectrón , Dicistroviridae/genética , Kluyveromyces/metabolismo , Modelos Moleculares , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mensajero/ultraestructura , ARN de Transferencia/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , ARN Viral/ultraestructura , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Cotranslational protein translocation is a universally conserved process for secretory and membrane protein biosynthesis. Nascent polypeptides emerging from a translating ribosome are either transported across or inserted into the membrane via the ribosome-bound Sec61 channel. Here, we report structures of a mammalian ribosome-Sec61 complex in both idle and translating states, determined to 3.4 and 3.9 Å resolution. The data sets permit building of a near-complete atomic model of the mammalian ribosome, visualization of A/P and P/E hybrid-state tRNAs, and analysis of a nascent polypeptide in the exit tunnel. Unprecedented chemical detail is observed for both the ribosome-Sec61 interaction and the conformational state of Sec61 upon ribosome binding. Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide. The high-resolution structure of the mammalian ribosome-Sec61 complex provides a valuable reference for future functional and structural studies.
Asunto(s)
Proteínas de la Membrana/química , Biosíntesis de Proteínas , Ribosomas/química , Animales , Cristalografía por Rayos X , Proteínas de la Membrana/metabolismo , Modelos Moleculares , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Canales de Translocación SEC , PorcinosRESUMEN
During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.
Asunto(s)
Factores Eucarióticos de Iniciación/metabolismo , Kluyveromyces/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Codón Iniciador , Microscopía por Crioelectrón , Modelos Moleculares , Datos de Secuencia Molecular , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Alineación de SecuenciaRESUMEN
Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.
Asunto(s)
Adenosina Trifosfatasas , Elementos Transponibles de ADN , Proteínas de Unión al ADN , Proteínas de Escherichia coli , ARN Bacteriano , Adenosina Trifosfatasas/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Elementos Transponibles de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Especificidad por Sustrato , Transposasas/metabolismoRESUMEN
Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.
Asunto(s)
Factor 1 Eucariótico de Iniciación , Factores Eucarióticos de Iniciación , ARN de Transferencia de Metionina , Subunidades Ribosómicas , Microscopía por Crioelectrón , Factor 1 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/genética , Humanos , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Subunidades Ribosómicas/química , Subunidades Ribosómicas/metabolismo , Imagen Individual de MoléculaRESUMEN
Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1-3. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.
Asunto(s)
Sistemas CRISPR-Cas , Elementos Transponibles de ADN , ADN Bacteriano/química , Vibrio cholerae/química , Microscopía por Crioelectrón , ADN Bacteriano/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Multimerización de Proteína , Estructura Cuaternaria de Proteína , ARN Bacteriano/química , Vibrio cholerae/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.
Asunto(s)
COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , ARN Mensajero/metabolismo , Ribosomas/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Humanos , Pandemias , Iniciación de la Cadena Peptídica Traduccional/genética , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Viral/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Proteínas no Estructurales Virales/genéticaRESUMEN
Colony collapse disorder (CCD) is a multi-faceted syndrome decimating bee populations worldwide, and a group of viruses of the widely distributed Dicistroviridae family have been identified as a causing agent of CCD. This family of viruses employs non-coding RNA sequences, called internal ribosomal entry sites (IRESs), to precisely exploit the host machinery for viral protein production. Using single-particle cryo-electron microscopy (cryo-EM), we have characterized how the IRES of Israeli acute paralysis virus (IAPV) intergenic region captures and redirects translating ribosomes toward viral RNA messages. We reconstituted two in vitro reactions targeting a pre-translocation and a post-translocation state of the IAPV-IRES in the ribosome, allowing us to identify six structures using image processing classification methods. From these, we reconstructed the trajectory of IAPV-IRES from the early small subunit recruitment to the final post-translocated state in the ribosome. An early commitment of IRES/ribosome complexes for global pre-translocation mimicry explains the high efficiency observed for this IRES. Efforts directed toward fighting CCD by targeting the IAPV-IRES using RNA-interference technology are underway, and the structural framework presented here may assist in further refining these approaches.
Asunto(s)
Biomimética , Dicistroviridae/fisiología , Sitios Internos de Entrada al Ribosoma/genética , Biosíntesis de Proteínas , ARN de Transferencia/genética , ARN Viral/genética , Ribosomas/metabolismo , Microscopía por Crioelectrón , Dicistroviridae/ultraestructura , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , ARN de Transferencia/ultraestructura , Ribosomas/ultraestructuraRESUMEN
Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a "dead-end" intermediate at 3.6 Šallowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states.
Asunto(s)
Factores Eucarióticos de Iniciación/metabolismo , Guanosina Trifosfato/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Sitios de Unión , Microscopía por Crioelectrón , Factores Eucarióticos de Iniciación/química , Factores Eucarióticos de Iniciación/genética , Hidrólisis , Mutagénesis Sitio-Dirigida , Unión Proteica , Ribosomas/metabolismo , Saccharomyces cerevisiaeRESUMEN
In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a ß-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.
Asunto(s)
Aminoácidos/deficiencia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , GTP Pirofosfoquinasa/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Estrés Fisiológico , Adenosina/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , GTP Pirofosfoquinasa/antagonistas & inhibidores , GTP Pirofosfoquinasa/genética , GTP Pirofosfoquinasa/ultraestructura , Regulación Bacteriana de la Expresión Génica , Guanosina Tetrafosfato/química , Guanosina Tetrafosfato/metabolismo , Redes y Vías Metabólicas , Modelos Moleculares , Fosforilación , Biosíntesis de Proteínas , Conformación Proteica , Estructura Terciaria de Proteína , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/ultraestructura , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/química , Ribosomas/ultraestructura , Sistemas de Mensajero SecundarioRESUMEN
During normal translation, the binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in the termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires the formation of two normally forbidden purine-purine base pairs. Here we determined the crystal structure at 3.1 Å resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNA(Ser) bound to the ΨAG stop codon in the A site. The ΨA base pair at the first position is accompanied by the formation of purine-purine base pairs at the second and third positions of the codon, which show an unusual Watson-Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding centre to accommodate non-canonical base pairs.
Asunto(s)
Emparejamiento Base , Codón de Terminación/genética , Codón de Terminación/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Anticodón/química , Anticodón/genética , Anticodón/metabolismo , Secuencia de Bases , Codón de Terminación/química , Cristalografía por Rayos X , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Seudouridina/química , Seudouridina/genética , Seudouridina/metabolismo , ARN de Transferencia de Serina/química , ARN de Transferencia de Serina/genética , ARN de Transferencia de Serina/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Ribosomas/genéticaRESUMEN
Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression of many physiological transcripts. SMG-1 is a large kinase essential to NMD that phosphorylates Upf1, which seems to be the definitive signal triggering mRNA decay. However, the regulation of the kinase activity of SMG-1 remains poorly understood. Here, we reveal the three-dimensional architecture of SMG-1 in complex with SMG-8 and SMG-9, and the structural mechanisms regulating SMG-1 kinase. A bent arm comprising a long region of HEAT (huntington, elongation factor 3, a subunit of PP2A and TOR1) repeats at the N terminus of SMG-1 functions as a scaffold for SMG-8 and SMG-9, and projects from the C-terminal core containing the phosphatidylinositol 3-kinase domain. SMG-9 seems to control the activity of SMG-1 indirectly through the recruitment of SMG-8 to the N-terminal HEAT repeat region of SMG-1. Notably, SMG-8 binding to the SMG-1:SMG-9 complex specifically down-regulates the kinase activity of SMG-1 on Upf1 without contacting the catalytic domain. Assembly of the SMG-1:SMG-8:SMG-9 complex induces a significant motion of the HEAT repeats that is signaled to the kinase domain. Thus, large-scale conformational changes induced by SMG-8 after SMG-9-mediated recruitment tune SMG-1 kinase activity to modulate NMD.
Asunto(s)
Modelos Moleculares , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Fosfatidilinositol 3-Quinasas/genética , Proteínas Quinasas/genética , Multimerización de Proteína/fisiología , Proteínas Serina-Treonina Quinasas , Estructura Cuaternaria de Proteína , ARN Helicasas , Proteínas Recombinantes/metabolismo , Transactivadores/metabolismoRESUMEN
The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre.
Asunto(s)
Proteínas de Escherichia coli/química , ARN de Transferencia/química , Ribosomas/metabolismo , Factor de Transcripción ReIA/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study the divergence of these lectins after gene duplication. Expression profiling of the three homodimeric (prototype) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence of distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identify differential changes in the circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behaviour, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism.
Asunto(s)
Galectina 2/química , Galectinas/química , Animales , Pollos , Cristalografía por Rayos X , Galectina 1/química , Galectina 2/metabolismo , Galectinas/metabolismo , Humanos , Ligandos , Modelos Químicos , Unión Proteica , Multimerización de Proteína , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin-binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin-binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin-binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF-driven mitogenesis.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Heparina/metabolismo , Resonancia Magnética Nuclear Biomolecular , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Sitios de Unión , Línea Celular , Dimerización , Factores de Crecimiento de Fibroblastos/química , Factores de Crecimiento de Fibroblastos/genética , Heparitina Sulfato/metabolismo , Humanos , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , UltracentrifugaciónRESUMEN
SMG-9 is part of a protein kinase complex, SMG1C, which consists of the SMG-1 kinase, SMG-8 and SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and targets for degradation mRNAs harboring premature translation termination codons. Here, we have characterized SMG-9, showing that it comprises an N-terminal 180 residue intrinsically disordered region (IDR) followed by a well-folded C-terminal domain. Both domains are required for SMG-1 binding and the integrity of the SMG1C complex, whereas the C-terminus is sufficient to interact with SMG-8. In addition, we have found that SMG-9 assembles in vivo into SMG-9:SMG-9 and, most likely, SMG-8:SMG-9 complexes that are not constituents of SMG1C. SMG-9 self-association is driven by interactions between the C-terminal domains and surprisingly, some SMG-9 oligomers are completely devoid of SMG-1 and SMG-8. We propose that SMG-9 has biological functions beyond SMG1C, as part of distinct SMG-9-containing complexes. Some of these complexes may function as intermediates potentially regulating SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of IDRs could facilitate the transit of SMG-9 through several macromolecular complexes.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/química , Codón sin Sentido , Células HeLa , Humanos , Multimerización de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismoRESUMEN
Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases.
Asunto(s)
Aspirina/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Gentisatos/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Antiinflamatorios no Esteroideos/metabolismo , Unión Competitiva , Movimiento Celular , Diseño de Fármacos , Matriz Extracelular/metabolismo , Gentisatos/clasificación , Gentisatos/farmacología , Heparina/química , Ratones , Ratones Endogámicos BALB C , Mitógenos/química , Células 3T3 NIHRESUMEN
Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learningbased structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.
Asunto(s)
Aprendizaje Profundo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mapeo de Interacción de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferasas/química , Aciltransferasas/metabolismo , Segregación Cromosómica , Biología Computacional , Simulación por Computador , Reparación del ADN , Evolución Molecular , Recombinación Homóloga , Ligasas/química , Ligasas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Biosíntesis de Proteínas , Conformación Proteica , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
Taking control of the cellular apparatus for protein production is a requirement for virus progression. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5'-UTR of a viral RNA assembles a functional initiation complex via an uAUG intermediate. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. The structures and accompanying functional data illustrate the importance of 5'-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs.