RESUMO
Pontin/RUVBL1 and Reptin/RUVBL2 are DNA-dependent ATPases involved in numerous cellular processes and are essential components of chromatin remodeling complexes and transcription factor assemblies. However, their existence as monomeric and oligomeric forms with differential activity in vivo reflects their versatility. Using a biochemical approach, we have studied the role of the nucleosome core particle and histone N-terminal tail modifications in the assembly and enzymatic activities of Reptin/Pontin. We demonstrate that purified Reptin and Pontin form stable complexes with nucleosomes. The ATPase activity of Reptin/Pontin is modulated by acetylation and methylation of the histone H3 N terminus. In vivo, association of Reptin with the progesterone receptor gene promoter is concomitant with changes in H3 marks of the surrounding nucleosomes. Furthermore, the presence of H3 tail peptides regulates the monomer-oligomer transition of Reptin/Pontin. Proteins that are pulled down by monomeric Reptin/Pontin differ from those that can bind to hexamers. We propose that changes in the oligomeric status of Reptin/Pontin create a platform that brings specific cofactors close to gene promoters and loads regulatory factors to establish an active state of chromatin.
Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , DNA/química , DNA Helicases/química , DNA Helicases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histonas/química , Histonas/genética , Humanos , Dados de Sequência Molecular , Nucleossomos/química , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The TIP49a and TIP49b proteins belong to the family of AAA+ ATPases and play essential roles in vital processes such as transcription, DNA repair, snoRNP biogenesis, and chromatin remodeling. We report the crystal structure of a TIP49b hexamer and the comparative analysis of large-scale conformational flexibility of TIP49a, TIP49b, and TIP49a/TIP49b complexes using molecular modeling and molecular dynamics simulations in a water environment. Our results establish key principles of domain mobility that affect protein conformation and biochemical properties, including a mechanistic basis for the downregulation of ATPase activity upon protein hexamerization. These approaches, applied to the lik-TIP49b mutant reported to possess enhanced DNA-independent ATPase activity, help explain how a three-amino acid insertion remotely affects the structure and conformational dynamics of the ATP binding and hydrolysis pocket while uncoupling ATP hydrolysis from DNA binding. This might be similar to the effects of conformations adopted by TIP49 heterohexamers.
