RESUMO
The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains.
Assuntos
Adenosina Difosfato Ribose/química , Poli(ADP-Ribose) Polimerases/síntese química , Ubiquitina-Proteína Ligases/síntese química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Simulação por Computador , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Poli(ADP-Ribose) Polimerases/química , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Ubiquitina-Proteína Ligases/químicaRESUMO
Alpha-helical region substitution was applied to the SIAH1 and EL5 RING fingers. The Williams-Beuren syndrome transcription factor (WSTF) PHD_SIAH1 and WSTF PHD_EL5 RING fingers were created as the artificial ubiquitin-ligating enzyme (E3). These fingers possess E3 activities of mono-ubiquitination and poly-ubiquitination, respectively, with ubiquitin-conjugating enzyme (E2)-binding capabilities. Artificial E3s bind two zinc atoms and adopt a zinc-dependent ordered structure and ubiquitinate upon themselves without a substrate and a tag. Ubiquitination experiments using biotinylated ubiquitin showed that the WSTF PHD_EL5 RING finger is poly-ubiquitinated via residue Lys(63) of ubiquitin. Substitution of alpha-helical region might be applicable to various RING fingers with mono-ubiquitination or poly-ubiquitination.
Assuntos
Peptídeos/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Peptídeos/síntese química , Poliubiquitina/química , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Domínios RING Finger , Especificidade por Substrato , Fatores de Transcrição/síntese química , Enzimas de Conjugação de Ubiquitina/síntese química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/síntese química , Ubiquitina-Proteína Ligases/química , Ubiquitinação , Zinco/químicaRESUMO
The creation of the artificial RING finger as ubiquitin-ligating enzyme (E3) has been demonstrated. In this study, by the alpha-helical region substitution between the EL5 RING finger and the Williams-Beuren syndrome transcription factor (WSTF) PHD finger, the artificial E3 (WSTF PHD_RING finger) was newly created. The experiments of the chemical modification of residues Cys and the circular dichroism spectra revealed that the WSTF PHD_RING finger binds two zinc atoms and adopts the zinc-dependent ordered-structure. In the substrate-independent ubiquitination assay, the WSTF PHD_RING finger functions as E3 and was poly- or mono-ubiquitinated. The present strategy is very simple and convenient, and consequently it might be widely applicable to the creation of various artificial E3 RING fingers with the specific ubiquitin-conjugating enzyme (E2)-binding capability.