Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

Abstract

Missense mutations in park2, encoding the parkin protein, account for ϳ50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ϳ160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys 150 -Cys 169 and Cys 196 -His 215 consisting of CX 2 -3 CX 11 CX 2 C and CX 4 -6 CX 10 -16 -CX 2 (H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn 2؉ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease. Autosomal recessive juvenile Parkinson disease (ARJP)2 is a neurodegenerative disorder arising from the loss of dopaminergic neurons in the substantia nigra of the midbrain. ARJP is characterized by the onset of Parkinsonian symptoms such as tremors, rigidity, and bradykinesia. It is distinguished from the idiopathic form of Parkinson disease by the onset of symptoms, prior to the age of forty. The hereditary nature of ARJP implicates a number of mutations in the genes encoding the proteins parkin, PINK1, LRRK2, and DJ-1 as the cause of dopaminergic neurodegeneration (1-4). A variety of deletion, truncation, and point mutations distributed throughout the park2 gene, which encodes the protein parkin, have been reported in ARJP patients (1,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).Parkin functions as a ubiquitin ligase (E3) and belongs to a family of RBR (RING-between-RING) ubiquitin li...

Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart

“…The C-terminal RING box region consists of three domains termed RING1, RING2 and IBR (for in-between-RING) (Fig. 3d) 25 . Various FLAG-tagged human Parkin mutants were transfected to DOXtreated HeLa cells.…”

Selective escape of proteins from the mitochondria during mitophagy

“…We next examined whether the E3 activity of Parkin is also required for FKBP38 translocation. A mutation (T240R) in the RING1 domain of Parkin abolishes the E3 activity of the protein 13,26 . Treatment with CCCP for 24 h induced neither mitophagy nor FKBP38 translocation in MEFs expressing EGFP-Parkin(T240R), whereas cytochrome c was degraded and FKBP38 translocated to the ER in those expressing wild-type EGFP-Parkin (Fig.…”

Differential Interaction of the E3 Ligase Parkin with the Proteasomal Subunit S5a and the Endocytic Protein Eps15

“…Mutations in parkin account for ϳ50% of all ARJP cases. Parkin is a 465-residue multidomain E3 ligase comprising an N-terminal ubiquitin-like domain (Ubld) followed by a unique parkin-specific domain, two RING domains (RING0, RING1), an in-between RING (IBR) domain, and a C-terminal RING domain (RING2) (7,8). Mutations associated with ARJP are found throughout the parkin protein and have profound affects on the folding and functionality of the protein.…”