Mechanism and regulation of the Lys6-selective deubiquitinase USP30

Damaged mitochondria undergo mitophagy, a specialized form of autophagy that is initiated by the protein kinase PINK1 and the ubiquitin E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonizes Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both show a preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has not been well studied. Here we report crystal structures of human USP30 bound to monoubiquitin and Lys6-linked diubiquitin, which explain how USP30 achieves Lys6-linkage preference through unique ubiquitin binding interfaces. We assess the interplay between USP30, PINK1 and Parkin and show that distally phosphorylated ubiquitin chains impair USP30 activity. Lys6-linkage-specific affimers identify numerous mitochondrial substrates for this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into the architecture, activity and regulation of USP30, which will aid drug design against this and related enzymes.