RESUMO
SETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.
Assuntos
Histona-Lisina N-Metiltransferase/economia , Processamento de Proteína Pós-Traducional/genética , Especificidade por Substrato/genética , Sequência de Aminoácidos/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Lisina , Metilação , Mutação/genética , Peptídeos/genéticaRESUMO
Chromokinesins NOD and KID have similar DNA binding domains and functions during cell division, while their motor domain sequences show significant variations. It has been unclear whether these motors have the similar structure, chemistry, and microtubule interactions necessary to follow a similar mechanism of force generation. We used biochemical rate measurements, cosedimentation, and structural analysis to investigate the ATPase mechanisms of the NOD and KID core domains. These studies revealed that NOD and KID have different ATPase mechanisms, microtubule interactions, and catalytic domain structures. The ATPase cycles of NOD and KID have different rate-limiting steps. The ATPase rate of NOD was robustly stimulated by microtubules, and its microtubule affinity was weakened in all nucleotide-bound states. KID bound microtubules tightly in all nucleotide states and remained associated with the microtubule for more than 100 cycles of ATP hydrolysis before dissociating. The structure of KID was most like that of conventional kinesin (KIF5). Key differences in the microtubule binding region and allosteric communication pathway between KID and NOD are consistent with our biochemical data. Our results support the model in which NOD and KID utilize distinct mechanistic pathways to achieve the same function during cell division.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/genética , Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Cinesinas/química , Cinesinas/genética , Cinética , Microtúbulos/química , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Domínios ProteicosRESUMO
Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands. The three evolved groups showed remarkable differences in core composition, illustrating the effect of different selective pressures on the core. Changes in the core did not significantly alter protein stability, but were linked closely to changes in binding affinity and specificity. Structural analysis and molecular dynamics simulations revealed the structural basis for altered specificity. The evolved domains had significantly reduced core volumes, which in turn induced increased backbone flexibility. These motions were propagated from the core to the binding surface and induced significant conformational changes. These results show that alternative core packing and consequent allosteric modulation of binding interfaces could be used to engineer proteins with novel functions.
Assuntos
Regulação Alostérica/fisiologia , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Domínios de Homologia de src/fisiologia , Sequência de Aminoácidos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
α-N-terminal methylation of proteins is an important post-translational modification that is catalyzed by two different N-terminal methyltransferases, namely NTMT1 and NTMT2. Previous studies have suggested that NTMT1 is a tri-methyltransferase, whereas NTMT2 is a mono-methyltransferase. Here, we report the first crystal structures, to our knowledge, of NTMT2 in binary complex with S-adenosyl-L-methionine as well as in ternary complex with S-adenosyl-L-homocysteine and a substrate peptide. Our structural observations combined with biochemical studies reveal that NTMT2 is also able to di-/tri-methylate the GPKRIA peptide and di-methylate the PPKRIA peptide, otherwise it is predominantly a mono-methyltransferase. The residue N89 of NTMT2 serves as a gatekeeper residue that regulates the binding of unmethylated versus monomethylated substrate peptide. Structural comparison of NTMT1 and NTMT2 prompts us to design a N89G mutant of NTMT2 that can profoundly alter its catalytic activities and product specificities.
RESUMO
The tuberous sclerosis complex (TSC) is a negative regulator of mTOR complex 1, a signaling node promoting cellular growth in response to various nutrients and growth factors. However, several regulators in TSC signaling still await discovery and characterization. Using pulldown and MS approaches, here we identified the TSC complex member, TBC1 domain family member 7 (TBC1D7), as a binding partner for PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), a negative regulator of Akt kinase signaling. Most TBC domain-containing proteins function as Rab GTPase-activating proteins (RabGAPs), but the crystal structure of TBC1D7 revealed that it lacks residues critical for RabGAP activity. Sequence analysis identified a putative site for both Akt-mediated phosphorylation and 14-3-3 binding at Ser-124, and we found that Akt phosphorylates TBC1D7 at Ser-124. However, this phosphorylation had no effect on the binding of TBC1D7 to TSC1, but stabilized TBC1D7. Moreover, 14-3-3 protein both bound and stabilized TBC1D7 in a growth factor-dependent manner, and a phospho-deficient substitution, S124A, prevented this interaction. The crystal structure of 14-3-3ζ in complex with a phospho-Ser-124 TBC1D7 peptide confirmed the direct interaction between 14-3-3 and TBC1D7. The sequence immediately upstream of Ser-124 aligned with a canonical ß-TrCP degron, and we found that the E3 ubiquitin ligase ß-TrCP2 ubiquitinates TBC1D7 and decreases its stability. Our findings reveal that Akt activity determines the phosphorylation status of TBC1D7 at the phospho-switch Ser-124, which governs binding to either 14-3-3 or ß-TrCP2, resulting in increased or decreased stability of TBC1D7, respectively.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Transporte/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esclerose Tuberosa , Sítios de Ligação , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Ligação Proteica , Estabilidade Proteica , Serina , Ubiquitinação , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
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
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
The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.
Assuntos
Simulação por Computador , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Histonas/química , Células-Tronco Embrionárias Humanas/citologia , Humanos , Metilação , Complexo Repressor Polycomb 2/químicaRESUMO
SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways. The majority of SH3 domains studied thus far act by binding to proline-rich sequences in partner proteins, but a growing number of studies have revealed alternative recognition mechanisms. We have comprehensively surveyed the specificity landscape of human SH3 domains in an unbiased manner using peptide-phage display and deep sequencing. Based on â¼70,000 unique binding peptides, we obtained 154 specificity profiles for 115 SH3 domains, which reveal that roughly half of the SH3 domains exhibit non-canonical specificities and collectively recognize a wide variety of peptide motifs, most of which were previously unknown. Crystal structures of SH3 domains with two distinct non-canonical specificities revealed novel peptide-binding modes through an extended surface outside of the canonical proline-binding site. Our results constitute a significant contribution toward a complete understanding of the mechanisms underlying SH3-mediated cellular responses.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas/química , Proteínas/metabolismo , Análise de Sequência de Proteína/métodos , Animais , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas/genética , Domínios de Homologia de srcRESUMO
Selective inhibitors of protein lysine methyltransferases, including SET domain-containing protein 8 (SETD8), are highly desired, as only a fraction of these enzymes are associated with high-quality inhibitors. From our previously discovered SETD8 inhibitor, we developed a more potent analog and solved a cocrystal structure, which is the first crystal structure of SETD8 in complex with a small-molecule inhibitor. This cocrystal structure allowed the design of a covalent inhibitor of SETD8 (MS453), which specifically modifies a cysteine residue near the inhibitor binding site, has an IC50 value of 804 nM, reacts with SETD8 with near-quantitative yield, and is selective for SETD8 against 28 other methyltransferases. We also solved the crystal structure of the covalent inhibitor in complex with SETD8. This work provides atomic-level perspective on the inhibition of SETD8 by small molecules and will help identify high-quality chemical probes of SETD8.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Thioredoxin-interacting protein (TXNIP) is a multifunctional protein involved in diverse cellular processes such as cell proliferation and apoptosis. TXNIP stability is controlled by the ubiquitin-proteasome pathway, and the E3 ubiquitin ligase Itch directly interacts with TXNIP via PPxY motifs of TXNIP. In a previously published study, we have shown that phosphorylation of the PPxY tyrosyl residue switches TXNIP selectivity between different binding partners. Here, we describe that tyrosine-phosphorylated PPxY motifs also bind to SH2 domains of Vav2 and Src with dissociation constants around 10 µm and that phosphorylation is indispensable for these interactions as well. The crystal structure of the complex between a phosphorylated PPxY motif, and the SH2 domain of Vav2 reveals a conserved recognition mechanism.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Proto-Oncogênicas c-vav/química , Proteínas Proto-Oncogênicas c-vav/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Tirosina/química , Ubiquitina-Proteína Ligases/metabolismo , Domínios de Homologia de srcRESUMO
We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a K(d) of â¼100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.
Assuntos
Oligopeptídeos/farmacologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genética , Animais , Disponibilidade Biológica , Biotinilação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Regulação da Expressão Gênica/genética , Humanos , Isomerismo , Ligases , Masculino , Metilação , Camundongos , Modelos Moleculares , Complexo Repressor Polycomb 1/biossíntese , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
TXNIP (thioredoxin-interacting protein) negatively regulates the antioxidative activity of thioredoxin and participates in pleiotropic cellular processes. Its deregulation is linked to various human diseases, including diabetes, acute myeloid leukaemia and cardiovascular diseases. The E3 ubiquitin ligase Itch (Itchy homologue) polyubiquitinates TXNIP to promote its degradation via the ubiquitin-proteasome pathway, and this Itch-mediated polyubiquitination of TXNIP is dependent on the interaction of the four WW domains of Itch with the two PPxY motifs of TXNIP. However, the molecular mechanism of this interaction of TXNIP with Itch remains elusive. In the present study, we found that each of the four WW domains of Itch exhibited different binding affinities for TXNIP, whereas multivalent engagement between the four WW domains of Itch and the two PPxY motifs of TXNIP resulted in their strong binding avidity. Our structural analyses demonstrated that the third and fourth WW domains of Itch were able to recognize both PPxY motifs of TXNIP simultaneously, supporting a multivalent binding mode between Itch and TXNIP. Interestingly, the phosphorylation status on the tyrosine residue of the PPxY motifs of TXNIP serves as a molecular switch in its choice of binding partners and thereby downstream biological signalling outcomes. Phosphorylation of this tyrosine residue of TXNIP diminished the binding capability of PPxY motifs of TXNIP to Itch, whereas this phosphorylation is a prerequisite to the binding activity of TXNIP to SHP2 [SH2 (Src homology 2) domain-containing protein tyrosine phosphatase 2] and their roles in stabilizing the phosphorylation and activation of CSK (c-Src tyrosine kinase).
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Prolina/análogos & derivados , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação/fisiologia , Prolina/química , Prolina/genética , Prolina/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
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
Mismatch repair prevents the accumulation of erroneous insertions/deletions and non-Watson-Crick base pairs in the genome. Pathogenic mutations in the MLH1 gene are associated with a predisposition to Lynch and Turcot's syndromes. Although genetic testing for these mutations is available, robust classification of variants requires strong clinical and functional support. Here, the first structure of the N-terminus of human MLH1, determined by X-ray crystallography, is described. The structure shares a high degree of similarity with previously determined prokaryotic MLH1 homologs; however, this structure affords a more accurate platform for the classification of MLH1 variants.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Nucleares/química , Proteínas Recombinantes de Fusão/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Clonagem Molecular , Neoplasias Colorretais Hereditárias sem Polipose/genética , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Homologia Estrutural de ProteínaRESUMO
N(6)-methyladenosine (m(6)A) is the most abundant internal modification of nearly all eukaryotic mRNAs and has recently been reported to be recognized by the YTH domain family proteins. Here we present the crystal structures of the YTH domain of YTHDC1, a member of the YTH domain family, and its complex with an m(6)A-containing RNA. Our structural studies, together with transcriptome-wide identification of YTHDC1-binding sites and biochemical experiments, not only reveal the specific mode of m(6)A-YTH binding but also explain the preferential recognition of the GG(m(6)A)C sequences by YTHDC1.
Assuntos
Adenosina/análogos & derivados , Proteínas do Tecido Nervoso/química , Proteínas de Ligação a RNA/química , RNA/química , Adenosina/química , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Proteínas do Tecido Nervoso/metabolismo , RNA/metabolismo , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
The RNA polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeats (1-YSPTSPS-7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD-interaction domains (CIDs) with RNAPII CTD repeats phosphorylated at S2 and S7. Crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with RNAPII CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Neoplasias/química , Processamento de Proteína Pós-Traducional , RNA Polimerase II/química , Proteínas Repressoras/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Serina/químicaRESUMO
Pathogens can interfere with vital biological processes of their host by mimicking host proteins. The NS1 protein of the influenza A H3N2 subtype possesses a histone H3K4-like sequence at its carboxyl terminus and has been reported to use this mimic to hijack host proteins. However, this mimic lacks a free N-terminus that is essential for binding to many known H3K4 readers. Here we show that the double chromodomains of CHD1 adopt an 'open pocket' to interact with the free N-terminal amine of H3K4, and the open pocket permits the NS1 mimic to bind in a distinct conformation. We also explored the possibility that NS1 hijacks other cellular proteins and found that the NS1 mimic has access to only a subset of chromatin-associated factors, such as WDR5. Moreover, methylation of the NS1 mimic can not be reversed by the H3K4 demethylase LSD1. Overall, we thus conclude that the NS1 mimic is an imperfect histone mimic.
Assuntos
Histonas/metabolismo , Interações Hospedeiro-Patógeno , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Calorimetria , Cristalografia por Raios X , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Vírus da Influenza A Subtipo H3N2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
N(6)-Methyladenosine (m(6)A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m(6)A in single-stranded RNA. Here we report crystal structures of ALKBH5 in the presence of either its cofactors or the ALKBH5 inhibitor citrate. Catalytic assays demonstrate that the ALKBH5 catalytic domain can demethylate both single-stranded RNA and single-stranded DNA. We identify the TCA cycle intermediate citrate as a modest inhibitor of ALKHB5 (IC50, â¼488 µm). The structural analysis reveals that a loop region of ALKBH5 is immobilized by a disulfide bond that apparently excludes the binding of dsDNA to ALKBH5. We identify the m(6)A binding pocket of ALKBH5 and the key residues involved in m(6)A recognition using mutagenesis and ITC binding experiments.
Assuntos
Dioxigenases/química , Proteínas de Membrana/química , RNA/química , Adenosina/análogos & derivados , Adenosina/química , Adenosina/genética , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase , Sítios de Ligação , Catálise , Cristalografia por Raios X , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metilação , Mutagênese , Ligação Proteica , Estrutura Secundária de Proteína , RNA/genética , RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia.