Understanding the Pathogenicity of Parkin Catalytic Domain Mutants

Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinsons disease (PD). Parkin and the mitochondrial kinase PINK1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally characterized and their structural basis revealed, several pathogenic PD mutations found in the catalytic RING2 domain remain poorly understood. Here, we characterize two pathogenic RING2 mutants, T415N and P437L and shed light on the underlying structural causes. For this purpose, we use biochemical in vitro assays in combination with AlphaFold modeling. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. After determining the parkin minimal ubiquitin binding region, we show that both mutants display impaired binding to the ubiquitin molecule charged onto the E2 enzyme. Finally, we employ the most recent version of AlphaFold 3 to generate a structural model of the phospho-parkin/phospho-ubiquitin/ubiquitin-charged E2 complex. This model consolidates our findings and provides a structural understanding for the pathogenicity of these two parkin variants. A better understanding of the different PD mutations at the molecular level can pave the way for personalized treatments and the design of small molecule therapeutics for the treatment of PD.