RESUMO
The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.
Assuntos
Ligação Proteica , Humanos , Proteólise , Células HEK293 , Sondas Moleculares/química , Sondas Moleculares/metabolismo , RNA Helicases DEAD-box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Degrons , Receptores de Interleucina-17RESUMO
The mixed-lineage leukemia (MLL)-AF10 fusion oncoprotein recruits DOT1L to the homeobox A (HOXA) gene cluster through its octapeptide motif leucine zipper (OM-LZ), thereby inducing and maintaining the MLL-AF10-associated leukemogenesis. However, the recognition mechanism between DOT1L and MLL-AF10 is unclear. Here, we present the crystal structures of both apo AF10OM-LZ and its complex with the coiled-coil domain of DOT1L. Disruption of the DOT1L-AF10 interface abrogates MLL-AF10-associated leukemic transformation. We further show that zinc stabilizes the DOT1L-AF10 complex and may be involved in the regulation of the HOXA gene expression. Our studies may also pave the way for the rational design of therapeutic drugs against MLL-rearranged leukemia.
Assuntos
Transformação Celular Neoplásica/patologia , Metiltransferases , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide , Fatores de Transcrição , Cristalização , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/genética , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Zinco/químicaRESUMO
We herewith applied a priori a generic hit identification method (POEM) for difficult targets of known three-dimensional structure, relying on the simple knowledge of physicochemical and topological properties of a user-selected cavity. Searching for local similarity to a set of fragment-bound protein microenvironments of known structure, a point cloud registration algorithm is first applied to align known subpockets to the target cavity. The resulting alignment then permits us to directly pose the corresponding seed fragments in a target cavity space not typically amenable to classical docking approaches. Last, linking potentially connectable atoms by a deep generative linker enables full ligand enumeration. When applied to the WD40 repeat (WDR) central cavity of leucine-rich repeat kinase 2 (LRRK2), an unprecedented binding site, POEM was able to quickly propose 94 potential hits, five of which were subsequently confirmed to bind in vitro to LRRK2-WDR.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Simulação de Acoplamento Molecular , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Sítios de Ligação , Domínios Proteicos , Humanos , Ligantes , Ligação Proteica , Repetições WD40 , AlgoritmosRESUMO
α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides, respectively, and reveal that NTMT1 contains two characteristic structural elements (a ß hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.
Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Cristalização , Ativação Enzimática/genética , Técnicas de Silenciamento de Genes , Metilação , Metiltransferases/genética , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , S-Adenosil-Homocisteína/químicaRESUMO
Bromodomain adjacent to zinc finger domain protein 2A (BAZ2A) (also called transcription termination factor-1 interacting protein 5), a key component of the nucleolar remodeling complex, recruits the nucleolar remodeling complex to ribosomal RNA genes, leading to their transcriptional repression. In addition to its tandem plant homeodomain-bromodomain that is involved in binding to acetylated histone H4, BAZ2A also contains a methyl-CpG-binding domain (MBD)-like Tip5/ARBP/MBD (TAM) domain that shares sequence homology with the MBD. In contrast with the methyl-CpG-binding ability of the canonical MBD, the BAZ2A TAM domain has been shown to bind to promoter-associated RNAs of ribosomal RNA genes and promoter DNAs of other genes independent of DNA methylation. Nevertheless, how the TAM domain binds to RNA/DNA mechanistically remains elusive. Here, we characterized the DNA-/RNA-binding basis of the BAZ2A TAM domain by EMSAs, isothermal titration calorimetry binding assays, mutagenesis analysis, and X-ray crystallography. Our results showed that the TAM domain of BAZ2A selectively binds to dsDNA and dsRNA and that it binds to the backbone of dsDNA in a sequence nonspecific manner, which is distinct from the base-specific binding of the canonical MBD. Thus, our results explain why the TAM domain of BAZ2A does not specifically bind to mCG or TG dsDNA like the canonical MBD and also provide insights for further biological study of BAZ2A acting as a transcription factor in the future.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , RNA/metabolismo , Proteínas Cromossômicas não Histona/química , DNA/química , Metilação de DNA , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , RNA/químicaRESUMO
The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/química , Malária/genética , Plasmodium falciparum/química , Conformação Proteica , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalografia por Raios X , GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/ultraestrutura , Humanos , Cinética , Malária/parasitologia , Plasmodium falciparum/patogenicidade , Plasmodium falciparum/ultraestrutura , Ligação ProteicaRESUMO
The NSD proteins, namely NSD1, NSD2 and NSD3, are lysine methyltransferases, which catalyze mono- and di-methylation of histone H3K36. They are multi-domain proteins, including two PWWP domains (PWWP1 and PWWP2) separated by some other domains. These proteins act as potent oncoproteins and are implicated in various cancers. However the biological functions of these PWWP domains are still largely unknown. To better understand the functions of these proteins' PWWP domains, we cloned, expressed and purified all the PWWP domains of these NSD proteins to characterize their interactions with methylated histone peptides and dsDNA by quantitative binding assays and crystallographic analysis. Our studies indicate that all these PWWP domains except NSD1_PWWP1 bind to trimethylated H3K36, H3K79 peptides and dsDNA weakly. Our crystal structures uncover that the NDS3_PWWP2 and NSD2_PWWP1 domains, which hold an extremely long α-helix and α-helix bundle, respectively, need a conformation adjustment to interact with nucleosome.
Assuntos
DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , DNA/química , DNA/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Humanos , Lisina/química , Lisina/metabolismo , Metilação , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Homologia de Sequência de AminoácidosRESUMO
The gene mutated in individuals with Huntington's disease (HD) encodes the 348-kDa huntingtin (HTT) protein. Pathogenic HD CAG-expansion mutations create a polyglutamine (polyQ) tract at the N terminus of HTT that expands above a critical threshold of â¼35 glutamine residues. The effect of these HD mutations on HTT is not well understood, in part because it is difficult to carry out biochemical, biophysical, and structural studies of this large protein. To facilitate such studies, here we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprised both N- and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, and juvenile HD patients, as well as the more extreme polyQ expansions used in some HD tissue and animal models. Our expression system yielded milligram quantities of pure recombinant HTT protein, including many of the previously mapped post-translational modifications. We characterized both apo and HTT-HTT-associated protein 40 (HAP40) complex samples produced with this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful HTT complexes. We further demonstrate that these resources can produce sufficient material for protein-intensive experiments, such as small-angle X-ray scattering, providing biochemical insight into full-length HTT protein structure. The work outlined and the tools generated here lay a foundation for further biochemical and structural work on the HTT protein and for studying its functional interactions with other biomolecules.
Assuntos
Expressão Gênica , Proteína Huntingtina/genética , Mutação , Animais , Clonagem Molecular , Humanos , Proteína Huntingtina/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , SpodopteraRESUMO
The N-end rule pathway senses the N-terminal destabilizing residues of degradation substrates for the ubiquitin-proteasome system, whose integrity shields against various human syndromes including cancer and cardiovascular diseases. GID4, a subunit of the ubiquitin ligase GID complex, has been recently identified as the N-recognin of the new branch of the N-end rule pathway responsible for recognizing substrates bearing N-terminal proline residues (Pro/N-degrons). However, the molecular mechanism of GID4-mediated Pro/N-degron recognition remains largely unexplored. Here, we report the first crystal structures of human GID4 alone and in complex with various Pro/N-degrons. Our complex crystal structures, together with biophysical analyses, delineate the GID4-mediated Pro/N-degron recognition mechanism and substrate selection criteria for the Pro/N-end rule pathway. These mechanistic data on the Pro/N-recognin activity of GID4 will serve as a foundation to facilitate the identification of authentic physiological substrates as well as the development of inhibitors of therapeutic values for the Pro/N-end rule pathway.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Humanos , Mutação , Prolina/química , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteólise , Especificidade por Substrato , Ubiquitina/química , Ubiquitina-Proteína Ligases/químicaRESUMO
Cytosine methylation is a well-characterized epigenetic mark and occurs at both CG and non-CG sites in DNA. Both methylated CG (mCG)- and mCH (H = A, C, or T)-containing DNAs, especially mCAC-containing DNAs, are recognized by methyl-CpG-binding protein 2 (MeCP2) to regulate gene expression in neuron development. However, the molecular mechanism involved in the binding of methyl-CpG-binding domain (MBD) of MeCP2 to these different DNA motifs is unclear. Here, we systematically characterized the DNA-binding selectivities of the MBD domains in MeCP2 and MBD1-4 with isothermal titration calorimetry-based binding assays, mutagenesis studies, and X-ray crystallography. We found that the MBD domains of MeCP2 and MBD1-4 bind mCG-containing DNAs independently of the sequence identity outside the mCG dinucleotide. Moreover, some MBD domains bound to both methylated and unmethylated CA dinucleotide-containing DNAs, with a preference for the CAC sequence motif. We also found that the MBD domains bind to mCA or nonmethylated CA DNA by recognizing the complementary TG dinucleotide, which is consistent with an overlooked ligand of MeCP2, i.e. the matrix/scaffold attachment regions (MARs/SARs) with a consensus sequence of 5'-GGTGT-3' that was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCA- and hmCA-containing dsDNAs. In summary, our results suggest that in addition to mCG sites, unmethylated CA or TG sites also serve as DNA-binding sites for MeCP2 and other MBD-containing proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation.
Assuntos
Proteínas de Ligação a DNA/química , DNA/metabolismo , Endodesoxirribonucleases/química , Proteína 2 de Ligação a Metil-CpG/química , Fatores de Transcrição/química , Sítios de Ligação , Calorimetria/métodos , Cristalografia por Raios X , Citosina/química , Metilação de DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Regulação da Expressão Gênica , Guanina/química , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Mutagênese , Nucleotídeos/metabolismo , Domínios Proteicos , Timina/química , Fatores de Transcrição/genéticaRESUMO
The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.
Assuntos
Biologia Computacional , Conformação Proteica , Proteínas/ultraestrutura , Arabidopsis/química , Arabidopsis/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , Humanos , Modelos Moleculares , Proteínas/química , Proteínas/genéticaRESUMO
NADPH-dependent antioxidant pathways have a critical role in scavenging hydrogen peroxide (H2O2) produced by oxidative phosphorylation. Inadequate scavenging results in H2O2 accumulation and can cause disease. To measure NADPH/NADP(+) redox states, we explored genetically encoded sensors based on steady-state fluorescence anisotropy due to FRET (fluorescence resonance energy transfer) between homologous fluorescent proteins (homoFRET); we refer to these sensors as Apollo sensors. We created an Apollo sensor for NADP(+) (Apollo-NADP(+)) that exploits NADP(+)-dependent homodimerization of enzymatically inactive glucose-6-phosphate dehydrogenase (G6PD). This sensor is reversible, responsive to glucose-stimulated metabolism and spectrally tunable for compatibility with many other sensors. We used Apollo-NADP(+) to study beta cells responding to oxidative stress and demonstrated that NADPH is significantly depleted before H2O2 accumulation by imaging a Cerulean-tagged version of Apollo-NADP(+) with the H2O2 sensor HyPer.
Assuntos
Técnicas Biossensoriais/métodos , Glucosefosfato Desidrogenase/metabolismo , Células Secretoras de Insulina/metabolismo , NADP/química , Células Cultivadas , Polarização de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Processamento de Imagem Assistida por Computador , NADP/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo , Conformação ProteicaRESUMO
Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of ß-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and ß-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer's disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where ß-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36.
Assuntos
Antígenos CD36/metabolismo , Proteínas de Membrana Lisossomal/química , Modelos Moleculares , Animais , Células CHO , Cricetulus , Células HeLa , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Heterochromatin protein 1 (HP1), a highly conserved non-histone chromosomal protein in eukaryotes, plays important roles in the regulation of gene transcription. Each of the three human homologs of HP1 includes a chromoshadow domain (CSD). The CSD interacts with various proteins bearing the PXVXL motif but also with a region of histone H3 that bears the similar PXXVXL motif. The latter interaction has not yet been resolved in atomic detail. Here we demonstrate that the CSDs of all three human HP1 homologs have comparable affinities to the PXXVXL motif of histone H3. The HP1 C-terminal extension enhances the affinity, as does the increasing length of the H3 peptide. The crystal structure of the human HP1γ CSD (CSDγ) in complex with an H3 peptide suggests that recognition of H3 by CSDγ to some extent resembles CSD-PXVXL interaction. Nevertheless, the prolyl residue of the PXXVXL motif appears to play a role distinct from that of Pro in the known HP1ß CSD-PXVXL complexes. We consequently generalize the historical CSD-PXVXL interaction model and expand the search scope for additional CSD binding partners.
Assuntos
Proteínas Cromossômicas não Histona/química , Motivos de Aminoácidos , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Cristalografia por Raios X , Humanos , Domínios ProteicosRESUMO
RBBP1 is a retinoblastoma protein (pRb) binding protein acting as a repressor of gene transcription. RBBP1 is a multidomain protein including a chromo barrel domain, and its chromo barrel domain has been reported to recognize histone H4K20me3 weakly, and this binding is enhanced by the simultaneous binding of DNA. However, the molecular basis of this DNA-mediated histone binding by the chromo barrel domain of RBBP1 is unclear. Here we attempted to co-crystallize the chromo barrel domain of RBBP1 with either a histone H4K20me3 peptide alone or with both a histone H4K20me3 peptide and DNA, but only solved the peptide/DNA unbound crystal structure. Our structural analysis indicates that RBBP1 could interact with histone H4K20me3 similar to other histone binding chromo barrel domains, and the surface charge representation analysis of the chromo barrel domain of RBBP1 suggests that the chromo barrel domain of RBBP1 does not have a typical DNA binding surface, indicating that it might not bind to DNA. Consistently, our ITC assays also showed that DNA does not significantly enhance the histone binding ability of the chromo barrel domain of RBBP1.
Assuntos
DNA/química , DNA/ultraestrutura , Histonas/química , Histonas/ultraestrutura , Simulação de Acoplamento Molecular , Proteína 1 de Ligação ao Retinoblastoma/química , Proteína 1 de Ligação ao Retinoblastoma/ultraestrutura , Sítios de Ligação , Modelos Químicos , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.
Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Cromatina/química , Imunoprecipitação/métodos , Proteômica/métodos , Clonagem Molecular , Biologia Computacional/métodos , Escherichia coli/metabolismo , Células HEK293 , Humanos , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Espectrometria de Massas/métodos , Biblioteca de Peptídeos , Proteínas/química , Proteoma , Reprodutibilidade dos TestesRESUMO
Covalent modifications of histone N-terminal tails play a critical role in regulating chromatin structure and controlling gene expression. These modifications are controlled by histone-modifying enzymes and read out by histone-binding proteins. Numerous proteins have been identified as histone modification readers. Here we report the family-wide characterization of histone binding abilities of human CW domain-containing proteins. We demonstrate that the CW domains in ZCWPW2 and MORC3/4 selectively recognize histone H3 trimethylated at Lys-4, similar to ZCWPW1 reported previously, while the MORC1/2 and LSD2 lack histone H3 Lys-4 binding ability. Our crystal structures of the CW domains of ZCWPW2 and MORC3 in complex with the histone H3 trimethylated at Lys-4 peptide reveal the molecular basis of this interaction. In each complex, two tryptophan residues in the CW domain form the "floor" and "right wall," respectively, of the methyllysine recognition cage. Our mutation results based on ZCWPW2 reveal that the right wall tryptophan residue is essential for binding, and the floor tryptophan residue enhances binding affinity. Our structural and mutational analysis highlights the conserved roles of the cage residues of CW domain across the histone methyllysine binders but also suggests why some CW domains lack histone binding ability.
Assuntos
Histonas/química , Proteínas Nucleares/química , Substituição de Aminoácidos , Histonas/genética , Histonas/metabolismo , Humanos , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de ProteínaRESUMO
PR domain-containing protein 7 (PRDM7) is a primate-specific histone methyltransferase that is the result of a recent gene duplication of PRDM9. The two proteins are highly homologous, especially in the catalytic PR/SET domain, where they differ by only three amino acid residues. Here we report that PRDM7 is an efficient methyltransferase that selectively catalyzes the trimethylation of H3 lysine 4 (H3K4) both in vitro and in cells. Through selective mutagenesis we have dissected the functional roles of each of the three divergent residues between the PR domains of PRDM7 and PRDM9. These studies indicate that after a single serine to tyrosine mutation at residue 357 (S357Y), PRDM7 regains the substrate specificities and catalytic activities similar to its evolutionary predecessor, including the ability to efficiently methylate H3K36.
Assuntos
Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Substituição de Aminoácidos , Duplicação Gênica , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Metilação , Mutagênese , Mutação de Sentido Incorreto , Especificidade por SubstratoRESUMO
Histone acetylation plays an important role in chromatin dynamics and is associated with active gene transcription. This modification is written by acetyltransferases, erased by histone deacetylases and read out by bromodomain containing proteins, and others such as tandem PHD fingers of DPF3b. Here we report the high resolution crystal structure of the tandem PHD fingers of DPF3b in complex with an H3K14ac peptide. In the complex structure, the histone peptide adopts an α-helical conformation, unlike previously observed by NMR, but similar to a previously reported MOZ-H3K14ac complex structure. Our crystal structure adds to existing evidence that points to the α-helix as a natural conformation of histone tails as they interact with histone-associated proteins.
Assuntos
Proteínas de Ligação a DNA/química , Histonas/química , Fatores de Transcrição/química , Acetilação , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dedos de Zinco PHD , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-TraducionalRESUMO
N(6)-Methyladenosine (m(6)A) is the most abundant internal modification in RNA and is specifically recognized by YT521-B homology (YTH) domain-containing proteins. Recently we reported that YTHDC1 prefers guanosine and disfavors adenosine at the position preceding the m(6)A nucleotide in RNA and preferentially binds to the GG(m(6)A)C sequence. Now we systematically characterized the binding affinities of the YTH domains of three other human proteins and yeast YTH domain protein Pho92 and determined the crystal structures of the YTH domains of human YTHDF1 and yeast Pho92 in complex with a 5-mer m(6)A RNA, respectively. Our binding and structural data revealed that the YTH domain used a conserved aromatic cage to recognize m(6)A. Nevertheless, none of these YTH domains, except YTHDC1, display sequence selectivity at the position preceding the m(6)A modification. Structural comparison of these different YTH domains revealed that among those, only YTHDC1 harbors a distinctly selective binding pocket for the nucleotide preceding the m(6)A nucleotide.