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 approximately 50% 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 approximately 160-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.

FIGURE 1.

Auto-ubiquitination of GST-parkin and parkin. A, Western blot (left panel) using ubiquitin antibody showing the in vitro auto-ubiquitination of GST-parkin in the presence of E1, Ubc7, and ubiquitin (fourth lane). In the absence of either E1 or ubiquitin (first and second lanes) or GST-parkin (third lane) auto-ubiquitination is not observed. The SDS-PAGE gel of the auto-ubiquitination reaction (right panel) shows multiple ubiquitinated species above the GST-parkin band. B, anti-ubiquitin Western blot (left panel) showing in vitro auto-ubiquitination of parkin in the presence of E1, Ubc7, and ubiquitin (fourth lane). A lack of ubiquitin modification occurs in the absence of E1 or ubiquitin (first and second lanes) or parkin (third lane). SDS-PAGE gel of the auto-ubiquitination reaction (right panel) showing multiple ubiquitinated species above the parkin band.

FIGURE 2.

Limited proteolysis of parkin by V8 protease. A, Coomassie Blue-stained SDS-PAGE gel of parkin following incubation with V8 protease over 120 min. B, schematic representation of parkin and major fragments resulting from V8 limited proteolysis that are detected by LC-MS. The calculated and observed molecular masses of each fragment are indicated to the right. C, LC-MS spectrum of parkin in the absence of V8 protease. D, LC-MS spectrum of parkin incubated with V8 protease for 30 min. E, LC-MS spectrum of parkin following 120 min of incubation with V8 protease. All of the calculated masses were derived from a parent parkin protein that also included an N-terminal GSPGIPARPAATTLPVT leader sequence that remained after thrombin cleavage.

FIGURE 3.

Limited proteolysis of parkin by trypsin protease. A, Coomassie Blue-stained SDS-PAGE gel of parkin following incubation with trypsin protease over 120 min. B, schematic representation of parkin and major fragments resulting from trypsin limited proteolysis that are detected by LC-MS. The calculated and observed molecular masses of each fragment are indicated to the right. C, LC-MS spectrum of parkin in the absence of trypsin protease. D, LC-MS spectrum of parkin following 10 min of trypsin proteolysis. E, LC-MS spectrum of parkin after incubation with trypsin protease for 90 min. All of the calculated masses were derived from a parent parkin protein that also included an N-terminal GSPGIPARPAATTLPVT leader sequence that remained after thrombin cleavage.

FIGURE 4.

Multiple sequence alignment of the RING0 domain of representative parkin orthologs. The region selected was identified from limited proteolysis experiments. The alignment was produced using ClustalW and manually curated by Jalview. The potential zinc binding residues are highlighted in yellow, and residue numbering is in reference to the human parkin sequence.

FIGURE 5.

Stoichiometry of zinc binding to parkin. A, ESI-MS spectra showing native full-length parkin with a mass of 53676.0 ± 1.0 Da corresponding to full-length parkin plus eight Zn²⁺ ions. B, ESI-MS of partially denatured parkin showing eight different parkin species, each with a mass difference of ∼63.5 Da resulting from the mass difference expected for binding of a single Zn²⁺ ion and loss of two protons. C, ESI-MS of fully denatured parkin with a mass of 53169.8 ± 2.2 Da and no bound Zn²⁺ ions.

FIGURE 6.

Removal of Zn²⁺ causes loss of structure in parkin. CD spectra of full-length parkin (3 μm) in 5 mm Tris, 20 mm NaCl, and 1 mm dithiothreitol at pH 7.4. The spectra show full-length parkin in the absence (thick solid line), and presence of four (dashed line) and eight (dotted line) equivalents of EDTA. Increased addition of EDTA resulted in no further changes in the CD spectrum. Also shown is the spectrum of unfolded parkin in the presence of 6 m guanidine hydrochloride (thin solid line).

FIGURE 7.

Schematic diagram of the new domain architecture of parkin. The domain structure is based on limited proteolysis, sequence conservation, and mass spectral data. The RING0 domain is located to the N-terminal side of the RBR region. Single residue ARJP mutations within RING0 are indicated.