Mechanism of Ubiquitin Ligase Activation of Parkin by a Small Molecule Molecular Glue

Mutations in parkin and PINK1 cause early-onset Parkinson’s disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry were performed with the most active compound (BIO-2007817) to identify the binding site and mechanism of action. We determined the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins, enhancing the affinity of pUb for RING0 and promoting the displacement of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 rescues the activity of EOPD mutants, R42P and V56E, with defective Ubl domains. This study provides the basis for the design of improved parkin activators as potential therapeutics for EOPD.