RESUMO
NSD3 is a histone H3 methyltransferase that plays an important role in chromatin biology. A construct containing the methyltransferase domain encompassing residues Q1049-K1299 of human NSD3 was obtained and biochemical activity was demonstrated using histone as a substrate. Here we report the backbone HN, N, Cα, C', and side chain Cß assignments of the construct in complex with S-adenosyl-L-methionine (SAM). Based on these assignments, secondary structures of NSD3/SAM complex in solution were determined.
Assuntos
Coenzimas/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Domínios PR-SET , Sequência de Aminoácidos , HumanosRESUMO
We have previously characterised the histone lysine methyltransferase properties of PRDM9, a member of the PRDM family of putative transcriptional regulators. PRDM9 displays broad substrate recognition and methylates a range of histone substrates, including octamers, core histone proteins, and peptides. In the present study, we show that PRDM9 performs intramolecular automethylation on multiple lysine residues localised to a lysine-rich region on the post-SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain. PRDM9 automethylation is abolished by a single active-site mutation, C321P, also known to disrupt interactions with S-adenosylmethionine. We have taken an initial step towards tool compound generation through rational design of a substrate-mimic, peptidic inhibitor of PRDM9 automethylation. The discovery of automethylation in PRDM9 adds a new dimension to our understanding of PRDM9 enzymology.
Assuntos
Cisteína/química , Histona-Lisina N-Metiltransferase/química , Prolina/química , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Domínio Catalítico , Clonagem Molecular , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Cinética , Ligantes , Metilação , Camundongos , Modelos Moleculares , Mutação , Prolina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Domínio Catalítico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.
