RESUMEN
PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observed that phospho-ubiquitin can bind to two distinct sites on parkin, a high-affinity site on RING1 that controls parkin localization and a low-affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho-ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.
Asunto(s)
Proteínas Quinasas , Ubiquitina-Proteína Ligasas , Fosforilación/genética , Dominios Proteicos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Mutations in the ubiquitin ligase parkin are responsible for a familial form of Parkinson's disease. Parkin and the PINK1 kinase regulate a quality-control system for mitochondria. PINK1 phosphorylates ubiquitin on the outer membrane of damaged mitochondria, thus leading to recruitment and activation of parkin via phosphorylation of its ubiquitin-like (Ubl) domain. Here, we describe the mechanism of parkin activation by phosphorylation. The crystal structure of phosphorylated Bactrocera dorsalis (oriental fruit fly) parkin in complex with phosphorylated ubiquitin and an E2 ubiquitin-conjugating enzyme reveals that the key activating step is movement of the Ubl domain and release of the catalytic RING2 domain. Hydrogen/deuterium exchange and NMR experiments with the various intermediates in the activation pathway confirm and extend the interpretation of the crystal structure to mammalian parkin. Our results rationalize previously unexplained Parkinson's disease mutations and the presence of internal linkers that allow large domain movements in parkin.
Asunto(s)
Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Cristalografía por Rayos X , Activación Enzimática , Humanos , Proteínas de Insectos/genética , Modelos Moleculares , Mutación , Fosforilación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas , Tephritidae/genética , Tephritidae/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
In the version of this article initially published, RING2 in the schematic to the left in Fig. 1b was mislabeled as RING0. The error has been corrected in the HTML and PDF versions of the article.