Mechanism and regulation of the Lys6-selective deubiquitinase USP30

Damaged mitochondria undergo a specialised form of autophagy termed mitophagy, which is initiated by the ubiquitin kinase PINK1 and the E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonises Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both display an unusual preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has remained poorly studied. We here report crystal structures of human USP30 bound to mono- 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 of this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into USP30 architecture, activity, and regulation, which will aid drug design against this and related enzymes.