Extended Data Fig. 8: A regulatory role of the parkin Ubl–UPD linker.

a, b, E2 discharge assay resolved on a Coomassie stained SDS–PAGE gel (a) and quantified from band intensities (b) for phospho-parkin and phospho-parkin(R104A). This is representative of at least two independent experiments; for gel source data, see Supplementary Fig. 1. The mutation in the ACT element leads to a reduction in discharge activity, suggesting that the residue is required to dislodge RING2 from the parkin core. c, Parkin(R104A) is equally stable as wild-type parkin, in the unphosphorylated or phosphorylated form. Thermal denaturation experiments were performed as technical triplicate. d, Sequence detail of the Ubl–UPD linker, which contains the ACT element described here. In the ACT element as bound to phospho-parkin–phospho-ubiquitin, the positions of two annotated (in PhosphoSitePlus) parkin phosphorylation sites, Ser101 and Ser108, are resolved. Phosphorylation of Ser101 decreases parkin activity⁴⁰, which is probably explained by phosphorylation preventing phospho-Ubl and/or linker binding to the UPD. It is hence highly likely that phosphorylation of parkin on these residues provides additional layers of parkin regulation that remain to be uncovered in future work. As an example, parkin phosphorylation by PKA was recently reported to be a mechanism of parkin inhibition in beige-to-white adipocyte transition, although phosphorylation sites remained unclear⁴¹. Residues before the ACT element (amino acids 73–99) and after the ACT element (amino acids 109–142) are disordered in our structure. The last ordered residue, Ser108, is tantalizingly close to the REP binding site as well as to the phospho-ubiquitin binding pocket, but disorder suggests that clear binding sites for other conserved linker residues, in particular for the parkin GLAVIL motif, are not present. HDX-MS also does not reveal additional protection of the linker, even when the E2–Ub conjugate is bound, suggesting that the GLAVIL motif may not bind the E2 (Fig. 1d, Extended Data Figs. 2, 3c). On the other hand, there are at least three additional annotated phosphorylation sites, Ser116, Ser131 and Ser136^15,40,42,43, suggesting that the second part of the linker may also be regulated. Phosphorylation on these residues could change the ability of the disordered parts of the linker to interact with parkin in cis. For example, we would speculate that a phosphorylated Ser116 could for example, reach the phosphate binding pocket occupied by phospho-Ser65 of ubiquitin. Alternatively, the remaining Ubl–UPD linker may be important for substrate recruitment, or involved in other, PINK1-independent mechanisms of parkin activation.