Structural basis for the pathogenicity of parkin catalytic domain mutants

Abstract

Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinson's disease. Parkin and the mitochondrial kinase PTEN-induced kinase 1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally and structurally characterized, several Parkinson's disease mutations found in the catalytic Rcat domain of parkin remain poorly understood. Here, we characterize two pathogenic Rcat mutants, T415N and P437L. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. We determine the minimal ubiquitin-binding segment and show that both mutants display impaired binding of ubiquitin charged on the E2 enzyme. Finally, we use AlphaFold 3 to predict a model of the phospho-parkin:phospho-ubiquitin:ubiquitin-charged E2 complex. The model shows the repressor element of parkin and the N-terminal residues of the catalytic domain form a helix to position ubiquitin for transfer from the E2 to parkin. Our results rationalize the pathogenicity of the parkin mutations and deepen our understanding of the active parkin:E2∼Ub complex.

Keywords: AlphaFold; E3 ubiquitin ligase; PRKN; parkin; structural biology; ubiquitin.