RESUMO
Lipid metabolism has been associated with the cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) stimulator of interferon genes (STING) DNA-sensing pathway, but our understanding of how these signals are integrated into a cohesive immunometabolic program is lacking. Here, we have identified liver X receptor (LXR) agonists as potent inhibitors of STING signaling. We show that stimulation of lipid metabolism by LXR agonists specifically suppressed cyclic GMP-AMP (cGAMP)-STING signaling. Moreover, we developed cyclic dinucleotide-conjugated beads to biochemically isolate host effectors for cGAMP inhibition, and we found that LXR ligands stimulated the expression of sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A), which is a 2'3'-cGAMP-degrading enzyme. Results of crystal structures suggest that cGAMP analog induces dimerization of SMPDL3A, and the dimerization is critical for cGAMP degradation. Additionally, we have provided evidence that SMPDL3A cleaves cGAMP to restrict STING signaling in cell culture and mouse models. Our results reveal SMPDL3A as a cGAMP-specific nuclease and demonstrate a mechanism for how LXR-associated lipid metabolism modulates STING-mediated innate immunity.
Assuntos
Metabolismo dos Lipídeos , Nucleotidiltransferases , Animais , Camundongos , Receptores X do Fígado/metabolismo , Nucleotidiltransferases/metabolismo , DNA , Nucleotídeos Cíclicos/metabolismo , Imunidade InataRESUMO
A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from December 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an atomic level, we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural analysis identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analysed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
Assuntos
Betacoronavirus/química , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Receptores Virais/química , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes/imunologia , Betacoronavirus/metabolismo , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Evolução Molecular , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , SARS-CoV-2 , Sais/química , Alinhamento de Sequência , Água/análise , Água/químicaRESUMO
The Staphylococcal Bap proteins sense environmental signals (such as pH, [Ca2+ ]) to build amyloid scaffold biofilm matrices via unknown mechanisms. We here report the crystal structure of the aggregation-prone region of Staphylococcus aureus Bap which adopts a dumbbell-shaped fold. The middle module (MM) connecting the N-terminal and C-terminal lobes consists of a tandem of novel double-Ca2+ -binding motifs involved in cooperative interaction networks, which undergoes Ca2+ -dependent order-disorder conformational switches. The N-terminal lobe is sufficient to mediate amyloid aggregation through liquid-liquid phase separation and maturation, and subsequent biofilm formation under acidic conditions. Such processes are promoted by disordered MM at low [Ca2+ ] but inhibited by ordered MM stabilized by Ca2+ binding, with inhibition efficiency depending on structural integrity of the interaction networks. These studies illustrate a novel protein switch in pathogenic bacteria and provide insights into the mechanistic understanding of Bap proteins in modulation of functional amyloid and biofilm formation, which could be implemented in the anti-biofilm drug design.
Assuntos
Amiloide/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Cálcio/metabolismo , Agregação Celular/fisiologiaRESUMO
BACKGROUND: circular RNAs (circRNAs) have been reported to exert important effects in the progression of numerous cancers. However, the functions of circRNAs in intrahepatic cholangiocarcinoma (ICC) are still unclear. METHODS: circPCNXL2 (has_circ_0016956) were identified in paired ICC by circRNA microarray. Then, we assessed the biological functions of circPCNXL2 by CCK8, EdU, clone formation, transwell, wound healing assays, and xenograft models. RNA pull-down, mass spectrometry, and RNA immunoprecipitation (RIP) were applied to explore the interaction between cirrcPCNXL2 and serine-threonine kinase receptor-associated protein (STRAP). RNA pull-down, RIP and luciferase reporter assays were used to investigate the sponge functions of circPCNXL2. In the end, we explore the effects of circPCNXL2 and trametinib (a MEK1/2 inhibitor) in vivo. RESULTS: circPCNXL2 was upregulated in ICC tissues and cell lines, which promoted the proliferation and metastasis of ICC in vitro and in vivo. In terms of the mechanisms, circPCNXL2 could directly bind to STRAP and induce the interaction between STRAP and MEK1/2, resulting in the tumor promotion in ICC by activation of ERK/MAPK pathways. Besides, circPCNXL2 could regulate the expression of SRSF1 by sponging miR-766-3p and subsequently facilitated the growth of ICC. Finally, circPCNXL2 could partially inhibit the anti-tumor activity of trametinib in vivo. CONCLUSION: circPCNXL2 played a crucial role in the progression of ICC by interacting with STRAP to activate the ERK signaling pathway, as well as by modulating the miR-766-3p/SRSF1 axis. These findings suggest that circPCNXL2 may be a promising biomarker and therapeutic target for ICC.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , MicroRNAs , Humanos , RNA Circular/genética , Proliferação de Células/genética , Colangiocarcinoma/metabolismo , Transdução de Sinais , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias dos Ductos Biliares/metabolismo , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.
Assuntos
Legionella pneumophila/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Arginina/metabolismo , Proteínas de Bactérias , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Chaperonas Moleculares/metabolismo , NAD/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Ubiquitina/químicaRESUMO
NuA4 catalyzes the acetylation of nucleosomes at histone H4, which is a well-established epigenetic event, controlling many genomic processes in Saccharomyces cerevisiae. Here we report the crystal structures of the NuA4 core complex and a cryoelectron microscopy structure with the nucleosome. The structures show that the histone-binding pocket of the enzyme is rearranged, suggesting its activation. The enzyme binds the histone tail mainly through the target lysine residue, with a preference for a small residue at the -1 position. The complex engages the nucleosome at the dish face and orients its catalytic pocket close to the H4 tail to achieve selective acetylation. The combined data reveal a space-sequence double recognition mechanism of the histone tails by a modifying enzyme in the context of the nucleosome.
Assuntos
Histona Acetiltransferases/química , Histonas/química , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Acetilação , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Lisina/química , Lisina/metabolismo , Simulação de Acoplamento Molecular , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
N6-methyladenine (6mA) of DNA is an emerging epigenetic mark in the genomes of Chlamydomonas, Caenorhabditis elegans, and mammals recently. Levels of 6mA undergo drastic fluctuation and thus affect fertility during meiosis and early embryogenesis. Here, we showed three complex structures of 6mA demethylase C. elegans NMAD-1A, a canonical isoform of NMAD-1 (F09F7.7). Biochemical results revealed that NMAD-1A prefers 6mA Bubble or Bulge DNAs. Structural studies of NMAD-1A revealed an unexpected "stretch-out" conformation of its Flip2 region, a conserved element that is usually bent over the catalytic center to facilitate substrate base flipping in other DNA demethylases. Moreover, the wide channel between the Flip1 and Flip2 of the NMAD-1A explained the observed preference of NMAD-1A for unpairing substrates, of which the flipped 6mA was primed for catalysis. Structural analysis and mutagenesis studies confirmed that key elements such as carboxy-terminal domain (CTD) and hypothetical zinc finger domain (ZFD) critically contributed to structural integrity, catalytic activity, and nucleosome binding. Collectively, our biochemical and structural studies suggest that NMAD-1A prefers to regulate 6mA in the unpairing regions and is thus possibly associated with dynamic chromosome regulation and meiosis regulation.
Assuntos
Ácidos Nucleicos , Animais , Caenorhabditis elegans/genética , Meiose , DNA , Desmetilação , MamíferosRESUMO
Industrial fermentation applications typically require enzymes that exhibit high stability and activity at high temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade-off between stability and activity. This report describes a computational strategy to enhance both activity and thermal stability of the mesophilic organophosphate-degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled a library of features associated with the target properties for each residue and then prioritized candidate sites by hierarchical clustering. Subsequent in silico screening with multiple algorithms to simulate selective pressures yielded a subset of 23 candidate mutations. Iterative parallel screening of mutations that improved thermal stability and activity yielded, MPHase-m5b, which exhibited 13.3 °C higher Tm and 4.2 times higher catalytic activity than wild-type (WT) MPH over a wide temperature range. Systematic analysis of crystal structures, molecular dynamics (MD) simulations, and Quantum Mechanics/Molecular Mechanics (QM/MM) calculations revealed a wider entrance to the active site that increased substrate access with an extensive network of interactions outside the active site that reinforced αß/ßα sandwich architecture to improve thermal stability. This study thus provides an advanced, rational design framework to improve efficiency in engineering highly active, thermostable biocatalysts for industrial applications.
RESUMO
Salt bridges are important factors in maintaining the stability of proteins, and their contribution to protein folding has received much attention. Although the interaction energies, or stabilizing contributions, of individual salt bridges have been measured in various proteins, a systematic assessment of various types of salt bridges in a relatively uniform environment is still a valuable analysis. Here, we used a collagen heterotrimer as a host-guest platform to construct 48 heterotrimers with the same charge pattern. A variety of salt bridges were formed between the oppositely charged residues Lys, Arg, Asp, and Glu. The melting temperature (Tm) of the heterotrimers was measured with circular dichroism. The atomic structures of 10 salt bridges were shown in three x-ray crystals of heterotrimer. Molecular dynamics simulation based on the crystal structures indicated that strong, intermediate, and weak salt bridges have distinctive N-O distances. A linear regression model was used to predict the stability of heterotrimers with high accuracy (R2 = 0.93). We developed an online database to help readers understand how a salt bridge stabilizes collagen. This work will help us better understand the stabilizing mechanism of salt bridges in collagen folding and provide a new strategy to design collagen heterotrimers.
Assuntos
Colágeno , Simulação de Dinâmica Molecular , Colágeno/metabolismo , Dicroísmo Circular , Dobramento de Proteína , Temperatura , Termodinâmica , Sais/químicaRESUMO
COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/epidemiologia , Adaptação ao Hospedeiro , Vison/imunologia , Mutação , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Adulto , Idoso , Enzima de Conversão de Angiotensina 2/genética , Animais , Sítios de Ligação , COVID-19/imunologia , COVID-19/terapia , COVID-19/transmissão , COVID-19/virologia , Feminino , Humanos , Imunização Passiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Vison/virologia , Simulação de Dinâmica Molecular , Países Baixos/epidemiologia , Ligação Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Adulto Jovem , Soroterapia para COVID-19RESUMO
BACKGROUND: Cholangiocarcinoma (CHOL) is the second most common primary hepatic malignant tumor, following hepatocellular carcinoma (HCC). CHOL is highly aggressive and heterogeneous resulting in poor prognosis. The diagnosis and prognosis of CHOL has not improved in the past decade. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is reported to be associated with tumors, however, its role in CHOL has not been revealed. This study is mainly for exploring the prognostic values and potential function of ACSL4 in CHOL. METHODS: We investigated the expression level and prognostic value of ACSL4 in CHOL based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. TIMER2.0, TISIDB and CIBERSORT databases were utilized to assess the associations between ACSL4 and immune infiltration cells in CHOL. Single-cell sequencing data from GSE138709 was analyzed to study the expression of ACSL4 in different types of cells. ACSL4 co-expressed genes were analyzed by Linkedomics. Additionally, Western Blot, qPCR, EdU assay, CCK8 assay, transwell assay and wound healing assay were performed to further confirm the roles of ACSL4 in the pathogenesis of CHOL. RESULTS: We found that the level of ACSL4 was higher in CHOL and it was correlated with the diagnosis and prognosis of CHOL patients. Then, we observed that the infiltration level of immune cells was related to the level of ACSL4 in CHOL. Moreover, ACSL4 and its co-expressed genes were mainly enriched in metabolism-related pathway and ACSL4 is also a key pro-ferroptosis gene in CHOL. Finally, knockdown of ACSL4 could reverse the tumor-promoting effect of ACSL4 in CHOL. CONCLUSIONS: The current findings demonstrated ACSL4 may as a novel biomarker for CHOL patients, which might regulate immune microenvironment and metabolism resulting in poor prognosis.
Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Colangiocarcinoma , Neoplasias Hepáticas , Humanos , Prognóstico , Colangiocarcinoma/genética , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Microambiente Tumoral/genéticaRESUMO
With the continuous development of society, people's life pressure is constantly increasing, and the mental health problems of college students are becoming increasingly prominent, bringing many challenges to their education and management. Universities should not only cultivate students' theoretical and professional knowledge and practical skills, but also attach importance to their mental health and effectively implement psychological education. Therefore, it is very necessary to develop and design a simple and effective student psychological evaluation system. As a new form of ideological and political transformation in universities in the era of big data, online ideological and political work has potential development space. It is necessary to carry out mental health education in universities, fully utilize online education forms, and improve ability of universities to repair mental health problems. Based on this, this system designs and implements software for typical image resolution based recognition and artificial intelligence. The use of B/S architecture in the development and use of. net technology and web server technology will enable more students to connect and use different terminals. In addition, an algorithm for image super-resolution recognition was proposed, which uses clustering convolution to improve residual blocks, improves modeling ability by extracting features on a larger scale, reduces the number of parameters to improve model calculation efficiency, and enables mental health educators and managers to work better. This article combines image super-resolution recognition technology with artificial intelligence technology to apply it to the process of psychological education in universities, thereby promoting the development of problem repair applications.
Assuntos
Inteligência Artificial , Estudantes , Humanos , Escolaridade , Educação em Saúde , Algoritmos , UniversidadesRESUMO
Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki â¼ 0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at â¼24 µM, and the potency of PF-3845 increased against an engineered strain Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.
Assuntos
Mycobacterium tuberculosis/enzimologia , Fenilalanina-tRNA Ligase/ultraestrutura , Conformação Proteica , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Sequência de Aminoácidos/genética , Antibacterianos/química , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Cinética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Fenilalanina-tRNA Ligase/química , Piperidinas/química , Piperidinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/enzimologia , Tuberculose Resistente a Múltiplos Medicamentos/genéticaRESUMO
Mycobacteria tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase (FAAH), was identified inhibiting Mtb PheRS at Ki ~0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modelling and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the Phe pocket while a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bi-substrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ~24 µM, and the potency of PF-3845 increased against Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.
RESUMO
The CRISPR-Cas systems, as exemplified by CRISPR-Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection. The CRISPR-Cpf1 system, a new class 2 CRISPR-Cas system, mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)(2+) ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Firmicutes/enzimologia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Sistemas CRISPR-Cas , Cristalografia por Raios X , Engenharia Genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Estabilidade de RNA , RNA Bacteriano/genética , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Especificidade por SubstratoRESUMO
Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The α/ß hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14-D3-ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/metabolismo , Lactonas/química , Lactonas/metabolismo , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Transdução de SinaisRESUMO
GapR is a nucleoid-associated protein that is an essential regulator of chromosome replication in the cell cycle model Caulobacter crescentus. Here, we demonstrate that free GapR is a homotetramer, but not a dimer as previously reported (Guo et al., Cell 175: 583-597, 2018). We have determined the crystal structure of GapR in complex with a 10-bp A-tract DNA, which has an open tetrameric conformation, different from the closed clamp conformation in the previously reported crystal structure of GapR/DNA complex. The free GapR adopts multiple conformations in dynamic exchange equilibrium, with the major conformation resembling the closed tetrameric conformation, while the open tetrameric conformation is a representative of minor conformers. As it is impossible for the circular genomic DNA to get into the central DNA binding tunnel of the major conformation, we propose that GapR initially binds DNA through the open conformation, and then undergoes structural rearrangement to form the closed conformation which fully encircles the DNA. GapR prefers to bind DNA with 10-bp consecutive A/T base pairs nonselectively (Kd â¼12 nM), while it can also bind GC-rich DNA sequence with a reasonable affinity of about 120 nM. Besides, our results suggest that GapR binding results in widening the minor groove of AT-rich DNA, instead of overtwisting DNA.
Assuntos
Proteínas de Bactérias/genética , DNA/genética , Conformação Molecular , Sequência de Aminoácidos/genética , Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/genética , Caulobacter crescentus/ultraestrutura , Divisão Celular/genética , DNA/ultraestrutura , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Conformação de Ácido Nucleico , Multimerização Proteica/genéticaRESUMO
The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, have been central to the study of solute transport. Using lipidic cubic phase crystallization and microfocus X-ray diffraction, we determined the structure of human GLUT3 in complex with D-glucose at 1.5 Å resolution in an outward-occluded conformation. The high-resolution structure allows discrimination of both α- and ß-anomers of D-glucose. Two additional structures of GLUT3 bound to the exofacial inhibitor maltose were obtained at 2.6 Å in the outward-open and 2.4 Å in the outward-occluded states. In all three structures, the ligands are predominantly coordinated by polar residues from the carboxy terminal domain. Conformational transition from outward-open to outward-occluded entails a prominent local rearrangement of the extracellular part of transmembrane segment TM7. Comparison of the outward-facing GLUT3 structures with the inward-open GLUT1 provides insights into the alternating access cycle for GLUTs, whereby the C-terminal domain provides the primary substrate-binding site and the amino-terminal domain undergoes rigid-body rotation with respect to the C-terminal domain. Our studies provide an important framework for the mechanistic and kinetic understanding of GLUTs and shed light on structure-guided ligand design.
Assuntos
Transportador de Glucose Tipo 3/química , Transportador de Glucose Tipo 3/metabolismo , Glucose/química , Glucose/metabolismo , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Cristalografia por Raios X , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 1/metabolismo , Humanos , Cinética , Ligantes , Maltose/química , Maltose/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rotação , Relação Estrutura-AtividadeRESUMO
One-quarter of the 28 types of natural collagen exist as heterotrimers. The oligomerization state of collagen affects the structure and mechanics of the extracellular matrix, providing essential cues to modulate biological and pathological processes. A lack of high-resolution structural information limits our mechanistic understanding of collagen heterospecific self-assembly. Here, the 1.77-Å resolution structure of a synthetic heterotrimer demonstrates the balance of intermolecular electrostatics and hydrogen bonding that affects collagen stability and heterospecificity of assembly. Atomistic simulations and mutagenesis based on the solved structure are used to explore the contributions of specific interactions to energetics. A predictive model of collagen stability and specificity is developed for engineering novel collagen structures.
Assuntos
Colágeno/química , Colágeno/metabolismo , Humanos , Simulação de Dinâmica Molecular , Multimerização Proteica , Estabilidade Proteica , Eletricidade Estática , TemperaturaRESUMO
The nucleoid-associated protein GapR found in Caulobacter crescentus is crucial for DNA replication, transcription, and cell division. Associated with overtwisted DNA in front of replication forks and the 3' end of highly-expressed genes, GapR can stimulate gyrase and topo IV to relax (+) supercoils, thus facilitating the movement of the replication and transcription machines. GapR forms a dimer-of-dimers structure in solution that can exist in either an open or a closed conformation. It initially binds DNA through the open conformation and then undergoes structural rearrangement to form a closed tetramer, with DNA wrapped in the central channel. Here, we show that the DNA binding domain of GapR (residues 1-72, GapRΔC17) exists as a dimer in solution and adopts the same fold as the two dimer units in the full-length tetrameric protein. It binds DNA at the minor groove and reads the spatial distribution of DNA phosphate groups through a lysine/arginine network, with a preference towards AT-rich overtwisted DNA. These findings indicate that the dimer unit of GapR has an intrinsic DNA binding preference. Thus, at the initial binding step, the open tetramer of GapR with two relatively independent dimer units can be more efficiently recruited to overtwisted regions.