ABSTRACT
Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86-130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.
Subject(s)
Enzyme Activation/genetics , Mitochondria/metabolism , Models, Molecular , Protein Kinases/chemistry , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Cloning, Molecular , Crystallization , Humans , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Scattering, Small Angle , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
Mutations in the parkin or PINK1 genes are the leading cause of the autosomal recessive form of Parkinson's disease. The gene products, the E3 ubiquitin ligase parkin and the serine/threonine kinase PINK1, are neuroprotective proteins, which act together in a mitochondrial quality control pathway. Here, we review the structure of parkin and mechanisms of its autoinhibition and function as a ubiquitin ligase. We present a model for the recruitment and activation of parkin as a key regulatory step in the clearance of depolarized or damaged mitochondria by autophagy (mitophagy). We conclude with a brief overview of other functions of parkin and considerations for drug discovery in the mitochondrial quality control pathway.
Subject(s)
Parkinson Disease/enzymology , Ubiquitin-Protein Ligases/physiology , Amino Acid Sequence , Animals , Humans , Mitochondria/enzymology , Mitophagy , Molecular Sequence Data , Neurons/enzymology , Parkinson Disease/pathology , Protein Transport , Ubiquitin-Protein Ligases/chemistry , UbiquitinationABSTRACT
Mutations in the PARK2 (parkin) gene are responsible for an autosomal recessive form of Parkinson's disease. The parkin protein is a RING-in-between-RING E3 ubiquitin ligase that exhibits low basal activity. We describe the crystal structure of full-length rat parkin. The structure shows parkin in an autoinhibited state and provides insight into how it is activated. RING0 occludes the ubiquitin acceptor site Cys(431) in RING2, whereas a repressor element of parkin binds RING1 and blocks its E2-binding site. Mutations that disrupted these inhibitory interactions activated parkin both in vitro and in cells. Parkin is neuroprotective, and these findings may provide a structural and mechanistic framework for enhancing parkin activity.
Subject(s)
Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Animals , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Mutation , Parkinson Disease , Parkinsonian Disorders , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Rats , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Zinc FingersABSTRACT
A new sesterterpene (1), a new norsesterterpene (3), and two new norditerpenes (4, 5) were isolated from the aerial parts of Salvia sahendica, together with 12 known compounds, comprising a sesterterpene, a sesquiterpene, a diterpene, two triterpenes, two steroidal compounds, and five flavonoids. The structures of the new compounds were established by spectroscopic data interpretation, and in the case of 4, its structure was confirmed by single-crystal X-ray analysis.