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1.
Mol Cell ; 81(1): 88-103.e6, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33220178

RESUMO

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.


Assuntos
Acetamidas/química , Cicloexilaminas/química , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Regulação Alostérica , Animais , Sítios de Ligação , Células CHO , Cricetulus , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Células HeLa , Humanos , Fosforilação
2.
Chembiochem ; 22(1): 120-123, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-32815262

RESUMO

Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.


Assuntos
Aminoácidos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Aminoácidos/química , Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Antígenos/química , Sítios de Ligação , Halogenação , Modelos Moleculares
3.
Cell Chem Biol ; 28(4): 475-486.e8, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33296667

RESUMO

The translation inhibitor rocaglamide A (RocA) has shown promising antitumor activity because it uniquely clamps eukaryotic initiation factor (eIF) 4A onto polypurine RNA for selective translational repression. As eIF4A has been speculated to be a unique target of RocA, alternative targets have not been investigated. Here, we reveal that DDX3 is another molecular target of RocA. Proximity-specific fluorescence labeling of an O-nitrobenzoxadiazole-conjugated derivative revealed that RocA binds to DDX3. RocA clamps the DDX3 protein onto polypurine RNA in an ATP-independent manner. Analysis of a de novo-assembled transcriptome from the plant Aglaia, a natural source of RocA, uncovered the amino acid critical for RocA binding. Moreover, ribosome profiling showed that because of the dominant-negative effect of RocA, high expression of eIF4A and DDX3 strengthens translational repression in cancer cells. This study indicates that sequence-selective clamping of DDX3 and eIF4A, and subsequent dominant-negative translational repression by RocA determine its tumor toxicity.


Assuntos
Benzofuranos/farmacologia , RNA Helicases DEAD-box/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Benzofuranos/química , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Inibidores Enzimáticos/química , Fator de Iniciação 4A em Eucariotos/metabolismo , Feminino , Humanos , Masculino , Modelos Moleculares , Conformação Molecular
4.
Biochemistry ; 59(13): 1299-1308, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32200625

RESUMO

The eukaryotic translation initiation factor eIF2 is a GTPase, which brings the initiator Met-tRNAi to the ribosome as the eIF2-GTP·Met-tRNAi ternary complex (TC). TC regeneration is catalyzed by the guanine nucleotide exchange factor (GEF) eIF2B. eIF2 phosphorylation by several stress-induced kinases converts it into a competitive inhibitor of eIF2B. Inhibition of eIF2B activity lowers cellular TC concentrations, which in turn triggers the integrated stress response (ISR). Depending on its degree of activation and duration, the ISR protects the cell from the stress or can itself induce apoptosis. ISR dysregulation is a causative factor in the pathology of multiple neurodegenerative disorders, while ISR inhibitors are neuroprotective. The realization that eIF2B is a promising therapeutic target has triggered significant interest in its structure and its mechanisms of action and regulation. Recently, four groups published the cryo-electron microscopy structures of eIF2B with its substrate eIF2 and/or its inhibitor, phosphorylated eIF2 [eIF2(α-P)]. While all three structures of the nonproductive eIF2B·eIF2(α-P) complex are similar to each other, there is a sharp disagreement between the published structures of the productive eIF2B·eIF2 complex. One group reports a structure similar to that of the nonproductive complex, whereas two others observe a vastly different eIF2B·eIF2 complex. Here, we discuss the recent reports on the structure, function, and regulation of eIF2B; the preclinical data on the use of ISR inhibitors for the treatment of neurodegenerative disorders; and how the new structural and biochemical information can inform and influence the use of eIF2B as a therapeutic target.


Assuntos
Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/metabolismo , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/genética , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fosforilação , Ligação Proteica , Conformação Proteica , Ribossomos/genética , Ribossomos/metabolismo , Estresse Fisiológico
5.
J Neurochem ; 154(1): 25-40, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31587290

RESUMO

Vanishing white matter disease (VWM) is an autosomal recessive neurological disorder caused by mutation(s) in any subunit of eukaryotic translation initiation factor 2B (eIF2B), an activator of translation initiation factor eIF2. VWM occurs with mutation of the genes encoding eIF2B subunits (EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5). However, little is known regarding the underlying pathogenetic mechanisms or how to treat patients with VWM. Here we describe the identification and detailed analysis of a new spontaneous mutant mouse harboring a point mutation in the Eif2b5 gene (p.Ile98Met). Homozygous Eif2b5I98M mutant mice exhibited a small body, abnormal gait, male and female infertility, epileptic seizures, and a shortened lifespan. Biochemical analyses indicated that the mutant eIF2B protein with the Eif2b5I98M mutation decreased guanine nucleotide exchange activity on eIF2, and the level of the endoplasmic reticulum stress marker activating transcription factor 4 was elevated in the 1-month-old Eif2b5I98M brain. Histological analyses indicated up-regulated glial fibrillary acidic protein immunoreactivity in the astrocytes of the Eif2b5I98M forebrain and translocation of Bergmann glia in the Eif2b5I98M cerebellum, as well as increased mRNA expression of an endoplasmic reticulum stress marker, C/EBP homologous protein. Disruption of myelin and clustering of oligodendrocyte progenitor cells were also indicated in the white matter of the Eif2b5I98M spinal cord at 8 months old. Our data show that Eif2b5I98M mutants are a good model for understanding VWM pathogenesis and therapy development. Cover Image for this issue: doi: 10.1111/jnc.14751.


Assuntos
Modelos Animais de Doenças , Fator de Iniciação 2B em Eucariotos/genética , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Neuroglia/patologia , Animais , Encéfalo/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação Puntual
6.
Science ; 364(6439): 495-499, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31048492

RESUMO

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.


Assuntos
Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Estresse Fisiológico , Motivos de Aminoácidos , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/metabolismo , Humanos , Fosforilação
7.
Mol Cell ; 74(6): 1205-1214.e8, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31080011

RESUMO

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.


Assuntos
Fatores de Iniciação em Eucariotos/química , Hepacivirus/genética , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Hepacivirus/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Sítios Internos de Entrada Ribossomal , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
8.
Mol Cell ; 73(4): 738-748.e9, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30595437

RESUMO

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.


Assuntos
Benzofuranos/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/metabolismo , RNA/metabolismo , Ribossomos/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Aglaia/química , Aglaia/genética , Aglaia/metabolismo , Substituição de Aminoácidos , Benzofuranos/química , Benzofuranos/isolamento & purificação , Benzofuranos/farmacologia , Sítios de Ligação , Resistência a Medicamentos/genética , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/genética , Células HEK293 , Humanos , Modelos Moleculares , Estrutura Molecular , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Domínios e Motivos de Interação entre Proteínas , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/isolamento & purificação , Inibidores da Síntese de Proteínas/farmacologia , RNA/química , Ribossomos/química , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Relação Estrutura-Atividade
9.
Sci Rep ; 8(1): 17435, 2018 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-30487538

RESUMO

Eukaryotic mRNA has a cap structure and a poly(A) tail at the 5' and 3' ends, respectively. The cap structure is recognized by eIF (eukaryotic translation initiation factor) 4 F, while the poly(A) tail is bound by poly(A)-binding protein (PABP). PABP has four RNA recognition motifs (RRM1-4), and RRM1-2 binds both the poly(A) tail and eIF4G component of eIF4F, resulting in enhancement of translation. Here, we show that PABP interacts with the 40S and 60S ribosomal subunits dynamically via RRM2-3 or RRM3-4. Using a reconstituted protein expression system, we demonstrate that wild-type PABP activates translation in a dose-dependent manner, while a PABP mutant that binds poly(A) RNA and eIF4G, but not the ribosome, fails to do so. From these results, functional significance of the interaction of PABP with the ribosome is discussed.


Assuntos
Proteínas de Ligação a Poli(A)/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Fator de Iniciação 4F em Eucariotos , Fator de Iniciação 4G em Eucariotos/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Ribossomos/química , Relação Estrutura-Atividade
10.
Epigenetics ; 13(4): 410-431, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30080437

RESUMO

The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.


Assuntos
Azepinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Histonas/química , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Triazóis/farmacologia , Acetilação/efeitos dos fármacos , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Modelos Moleculares , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição , Sítio de Iniciação de Transcrição/efeitos dos fármacos
11.
ACS Synth Biol ; 7(3): 801-806, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29480717

RESUMO

The genetic code in bacteria and animal cells has been expanded to incorporate novel amino acids into proteins. Recent efforts have enabled genetic code expansion in nematodes, flies, and mice, whereas such engineering is rare with industrially useful animals. In the present study, we engineered the silkworm Bombyx mori to synthesize silk fiber functionalized with azidophenylalanine. For this purpose, we developed a bacterial system to screen for B. mori phenylalanyl-tRNA synthetases with altered amino-acid specificity. We created four transgenic B. mori lines expressing the selected synthetase variants in silk glands, and found that two of them supported the efficient in vivo incorporation of azidophenylalanine into silk fiber. The obtained silk was bio-orthogonally reactive with fluorescent molecules. The results showed that genetic code expansion in an industrial animal can be facilitated by prior bacterial selection, to accelerate the development of silk fiber with novel properties.


Assuntos
Bombyx/genética , Código Genético , Seda/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Química Click , Fluorescência , Humanos , Fenilalanina/metabolismo
12.
PLoS One ; 13(2): e0193158, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29462206

RESUMO

Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-µl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.


Assuntos
Complexo Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Fragmentos Fab das Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Epitopos , Humanos , Modelos Moleculares , Conformação Proteica
13.
Biomolecules ; 7(1)2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28335556

RESUMO

The N¹-atom of guanosine at position 37 in transfer RNA (tRNA) is methylated by tRNA methyltransferase 5 (Trm5) in eukaryotes and archaea, and by tRNA methyltransferase D (TrmD) in bacteria. The resultant modified nucleotide m¹G37 positively regulates the aminoacylation of the tRNA, and simultaneously functions to prevent the +1 frameshift on the ribosome. Interestingly, Trm5 and TrmD have completely distinct origins, and therefore bear different tertiary folds. In this review, we describe the different strategies utilized by Trm5 and TrmD to recognize their substrate tRNAs, mainly based on their crystal structures complexed with substrate tRNAs.


Assuntos
RNA de Transferência/metabolismo , tRNA Metiltransferases/metabolismo , Aminoacilação , Catálise , Cristalografia por Raios X , Modelos Moleculares , RNA de Transferência/química , Especificidade por Substrato , tRNA Metiltransferases/química
14.
Brain Nerve ; 69(1): 45-50, 2017 Jan.
Artigo em Japonês | MEDLINE | ID: mdl-28126977

RESUMO

CACH/VWM (childhood ataxia with central nervous system hypomyelination/vanishing white matter) disease is caused by mutations in the genes encoding the subunits of eukaryotic initiation factor 2B (eIF2B), but its etiology is poorly understood. Here, we attempt to provide an explanation for the pathogenic mechanism of this disease, based on the crystal structure of eIF2B.


Assuntos
Fator de Iniciação 2B em Eucariotos/química , Leucoencefalopatias , Substância Branca/química , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Modelos Moleculares , Mutação , Estrutura Quaternária de Proteína
15.
FEBS J ; 284(6): 868-874, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27627185

RESUMO

Eukaryotic translation initiation factor 2B (eIF2B), a heterodecameric complex of two sets of the α, ß, γ, δ, and ε subunits, is the guanine nucleotide exchange factor (GEF) specific for eIF2, a heterotrimeric G protein consisting of the α, ß, and γ subunits. The eIF2 protein binds GTP on the γ subunits and delivers an initiator methionyl-tRNA (Met-tRNAiMet ) to the ribosome. The GEF activity of eIF2B is inhibited by stress-induced phosphorylation of Ser51 in the α subunit of eIF2, which leads to lower amounts of active eIF2 and a limited quantity of Met-tRNAiMet for the ribosome, resulting in global repression of translation. However, the structural mechanism of the GEF activity inhibition remained enigmatic, and therefore the three-dimensional structure of the entire eIF2B molecule had been awaited. Recently, we determined the crystal structure of Schizosaccharomyces pombe eIF2B. In this Structural Snapshot, we present the structural features of eIF2B and the mechanism underlying the GEF activity inhibition by the phosphorylation of eIF2α, elucidated from structure-based in vitro analyses.


Assuntos
Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Conformação Proteica , Relação Estrutura-Atividade , Sítios de Ligação , Cristalografia por Raios X , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosforilação , Ligação Proteica , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Ribossomos/química , Ribossomos/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética
16.
Protein Sci ; 26(2): 280-291, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27862552

RESUMO

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598-631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of ß1-α1-ß2-ß3-α2-ß4. Furthermore, a docking model based on NOESY measurements suggests that residues 607-616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix-helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull-down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix-helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145.


Assuntos
Simulação de Acoplamento Molecular , Dobramento de Proteína , Fatores de Processamento de RNA/química , Motivos de Aminoácidos , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo
17.
Nat Struct Mol Biol ; 23(10): 941-948, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27571175

RESUMO

Proteins with knotted configurations, in comparison with unknotted proteins, are restricted in conformational space. Little is known regarding whether knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. TrmD is a bacterial methyltransferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product, m1G37-tRNA, is essential for life and maintains protein-synthesis reading frames. Using an integrated approach of structural, kinetic, and computational analysis, we show that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot undergoes complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding, thereby stabilizing tRNA binding and allowing assembly of the active site. This work demonstrates new principles of knots as organized structures that capture the free energies of substrate binding and facilitate catalysis.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , RNA de Transferência/metabolismo , tRNA Metiltransferases/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Simulação de Dinâmica Molecular , Conformação Proteica , Dobramento de Proteína , RNA de Transferência/química , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/química
18.
J Struct Funct Genomics ; 17(1): 33-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27023709

RESUMO

Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.


Assuntos
Fator de Iniciação 2B em Eucariotos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cromatografia em Gel , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/genética , Expressão Gênica , Humanos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
19.
Nature ; 531(7592): 122-5, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26901872

RESUMO

Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, a heterotrimeric G protein consisting of α-, ß- and γ-subunits. eIF2B exchanges GDP for GTP on the γ-subunit of eIF2 (eIF2γ), and is inhibited by stress-induced phosphorylation of eIF2α. eIF2B is a heterodecameric complex of two copies each of the α-, ß-, γ-, δ- and ε-subunits; its α-, ß- and δ-subunits constitute the regulatory subcomplex, while the γ- and ε-subunits form the catalytic subcomplex. The three-dimensional structure of the entire eIF2B complex has not been determined. Here we present the crystal structure of Schizosaccharomyces pombe eIF2B with an unprecedented subunit arrangement, in which the α2ß2δ2 hexameric regulatory subcomplex binds two γε dimeric catalytic subcomplexes on its opposite sides. A structure-based in vitro analysis by a surface-scanning site-directed photo-cross-linking method identified the eIF2α-binding and eIF2γ-binding interfaces, located far apart on the regulatory and catalytic subcomplexes, respectively. The eIF2γ-binding interface is located close to the conserved 'NF motif', which is important for nucleotide exchange. A structural model was constructed for the complex of eIF2B with phosphorylated eIF2α, which binds to eIF2B more strongly than the unphosphorylated form. These results indicate that the eIF2α phosphorylation generates the 'nonproductive' eIF2-eIF2B complex, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control.


Assuntos
Fator de Iniciação 2B em Eucariotos/química , Schizosaccharomyces/química , Motivos de Aminoácidos , Sítios de Ligação , Biocatálise , Reagentes para Ligações Cruzadas/química , Cristalografia por Raios X , Fator de Iniciação 2B em Eucariotos/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
20.
Proc Natl Acad Sci U S A ; 112(31): E4197-205, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26183229

RESUMO

The deep trefoil knot architecture is unique to the SpoU and tRNA methyltransferase D (TrmD) (SPOUT) family of methyltransferases (MTases) in all three domains of life. In bacteria, TrmD catalyzes the N(1)-methylguanosine (m(1)G) modification at position 37 in transfer RNAs (tRNAs) with the (36)GG(37) sequence, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. The m(1)G37-modified tRNA functions properly to prevent +1 frameshift errors on the ribosome. Here we report the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. Our structural analysis revealed the mechanism by which TrmD binds the substrate tRNA in an AdoMet-dependent manner. The trefoil-knot center, which is structurally conserved among SPOUT MTases, accommodates the adenosine moiety of AdoMet by loosening/retightening of the knot. The TrmD-specific regions surrounding the trefoil knot recognize the methionine moiety of AdoMet, and thereby establish the entire TrmD structure for global interactions with tRNA and sequential and specific accommodations of G37 and G36, resulting in the synthesis of m(1)G37-tRNA.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Haemophilus influenzae/enzimologia , RNA de Transferência/metabolismo , Thermotoga maritima/enzimologia , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Sequência de Aminoácidos , Anticódon/genética , Sequência de Bases , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Guanina/metabolismo , Cinética , Metilação , Modelos Moleculares , Dados de Sequência Molecular , RNA de Transferência/química , RNA de Transferência/genética , S-Adenosilmetionina , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato
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