Binding to serine 65‐phosphorylated ubiquitin primes Parkin for optimal PINK1‐dependent phosphorylation and activation

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser⁶⁵)—which lies within its ubiquitin‐like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser⁶⁵. How Ser⁶⁵‐phosphorylated ubiquitin (ubiquitin^{Phospho‐Ser65}) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin^{Phospho‐Ser65} binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser⁶⁵ by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site‐directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin^{Phospho‐Ser65}, thereby promoting Parkin Ser⁶⁵ phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser⁶⁵ phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin^{Phospho‐Ser65} to Parkin disrupts the interaction between the Ubl domain and C‐terminal region, thereby increasing the accessibility of Parkin Ser⁶⁵. Finally, purified Parkin maximally phosphorylated at Ser⁶⁵ in vitro cannot be further activated by the addition of ubiquitin^{Phospho‐Ser65}. Our results thus suggest that a major role of ubiquitin^{Phospho‐Ser65} is to promote PINK1‐mediated phosphorylation of Parkin at Ser⁶⁵, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho‐Ser⁶⁵‐binding pocket on the surface of Parkin that is critical for the ubiquitin^{Phospho‐Ser65} interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin^{Phospho‐Ser65}, which could aid in the development of Parkin activators that mimic the effect of ubiquitin^{Phospho‐Ser65}.