Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early‐onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK 1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N‐terminal ubiquitin‐like ( pU bl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho‐ubiquitin‐loaded C‐terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING 1/ IBR interface. Further, NMR and mass spectrometry experiments indicate the RING 0/ RING 2 interface is re‐modelled, remote from the E2 binding site, and this alters the reactivity of the RING 2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pU bl domain with the RING 0 domain and rearrange the location of the RING 2(Rcat) domain to drive parkin activity.