Extended Data Figure 10: PhosphoUbl-induced ‘opening’ of PARKIN relies on a phosphate pocket in the UPD.

a, A second scenario would be that the phosphorylated Ubl domain rebinds to PARKIN at an alternative site. We previously speculated that the UPD contains a phosphate-binding site that is lined by two AR-JP patient mutations, K161N and K211N, and also contains Arg163 (ref. 14). b, Full-length HsPARKIN mutants as indicated were phosphorylated in vitro with GST–PhPINK1, resolved on SDS–PAGE and western blotted using an anti-pSer65 PARKIN antibody (Abcam cat no. ab154995). These proteins were used in c. c, Ub-vinyl sulfone (Ub-VS) modification of the active site Cys431 of HsPARKIN and HsPARKIN mutants with and without phosphoUb from a time course experiment is assessed on Coomassie-stained gels. Ub-VS reacts with ‘open’ forms of PARKIN, and was previously shown to modify phospho-PARKIN but not phosphoUb-activated PARKIN⁷. Phospho-PARKIN-mediated opening depends on the phosphate pocket present in the UPD, since phospho-PARKIN K211N, K161N (two AR-JP patient mutations) and R163E abrogated or impaired modification by Ub-VS and do not appear to have an accessible catalytic Cys, while the phosphorylated phosphoUb-binding-deficient mutant K151E is readily modified. The experiment was performed two times with consistent results and gels have been collated from two different assays (indicated by the gap). d, PARKIN ubiquitination reactions in presence of E1, UBE2L3, ubiquitin or ubiquitin S65A, ATP and GST-TcPINK1 for 2 h. PARKIN is activated by phosphoUb or by PARKIN Ubl phosphorylation in absence of phosphoUb (with ubiquitin S65A) (lanes 2/3). Mutants in the UPD phosphate pocket can still be activated by phosphoUb (albeit not to the same extent) (lanes 5, 8, 11) but are inactive when the Ubl domain is phosphorylated (lanes 6, 9, 12). This could suggest that the phosphoUbl binds back to the PARKIN UPD pocket. However, we cannot exclude the possibility that, for example, the linker between the Ubl and the UPD plays a more active role in PARKIN activation. The experiment was performed three times with consistent results. Molecular weight markers are in kDa for b–d.