title: Isolated RING2 domain of parkin is sufficient for E2-dependent E3 ligase activity.
abstract: The E3 ubiquitin ligase activity of the parkin protein is implicated in playing a protective role against neurodegenerative disorders including Parkinson's, Huntington's, and Alzheimer's diseases. Parkin has four zinc-containing domains: RING0, RING1, IBR (in-between ring), and RING2. Mutational analysis of full-length parkin suggests that the C-terminal RING2 domain contains the catalytic core. Here, a catalytically competent recombinant RING2 containing an N-terminal GB1 solubility peptide is described. In cell-free in vitro ubiqitination reactions, the RING2 construct catalyzes the transfer of ubiquitin from the E2 enzyme UbcH7 to the attached GB1 tag. This intramolecular autoubiquitination reaction indicates that (a) ubiquitination by RING2 can occur in the absence of other parkin domains and (b) UbcH7 can interact directly with RING2 to transfer its bound ubiquitin. Mass spectrometry identified sites of mono- and diubiquitin attachment to two surface-exposed lysine residues (Lys24 and Lys39) on the GB1 peptide. The sites of diubiquitination involved Lys11 and Lys48 linkages, which have been identified as general signals for proteasome degradation. Cleaving the linker between the GB1 tag and RING2 resulted in loss of ubiquitination activity, indicating that the substrate must be tethered to RING2 for proper presentation to the active site. Atomic absorption spectrometry and selective mutation of zinc ligands indicated that only one of the two zinc binding sites on RING2, the N-terminal site, needs to be occupied by zinc for expression of ubiquitination activity. This is consistent with the hypothesis that the second, C-terminal, zinc binding site on RING2 has a regulatory rather than a catalytic function.
authors: Departments of Molecular Biosciences and ‡Chemistry, §Analytical Proteomics Laboratory, The University of Kansas , Lawrence, Kansas 66044, United States.
authors: Carolyn A
authors: CA
authors: Rankin
authors: Nadezhda A
authors: NA
authors: Galeva
authors: KyeongMin
authors: K
authors: Bae
authors: Mirza Nayyar
authors: MN
authors: Ahmad
authors: Travis M
authors: TM
authors: Witte
authors: Mark L
authors: ML
authors: Richter
mesh_terms: D001665:Binding Sites / Q000235:genetics; D006801:Humans; D017434:Protein Structure, Tertiary; D013054:Spectrophotometry, Atomic; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D015032:Zinc / Q000378:metabolism
chemical_list: C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D015032:Zinc