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.