histidine His433, which accepts a proton from Cys431 to activate it, and glutamate Glu444, which is involved in autoubiquitination. Together these form the catalytic triad, whose assembly is required for parkin activation. Parkin also contains an N-terminal Ub-like domain (Ubl) for specific substrate recognition, a unique RING0 domain and a repressor (REP) region that tonically suppresses ligase activity. Under resting conditions, the tightly coiled conformation of parkin renders it inactive, as access to the catalytic RING2 residue is sterically blocked by RING0, while the E2 binding domain on RING1 is occluded by Ubl and REP. Activating stimuli disrupt these interdomain interactions and induce parkin to collapse along the RING1-RING0 interface. The active site of RING2 is drawn towards E2-Ub bound to RING1, facilitating formation of the Ub-thioester intermediate. Parkin activation requires phosphorylation of serine Ser65 in Ubl by serine/threonine kinase, PINK1. Addition of a charged phosphate destabilises hydrophobic interactions between Ubl and neighbouring subregions, reducing autoinhibitory effects of this N-terminus domain. Ser65Ala missense mutations were found to ablate Ub-parkin binding whilst inhibiting parkin recruitment to damaged mitochondria. PINK1 also phosphorylates Ub at Ser65, accelerating its discharge from E2 and enhancing its affinity for parkin. Although structural changes following phosphorylation are uncertain, crystallisation of parkin revealed a cationic pocket in RING0 formed by lysine and arginine residues Lys161, Arg163 and Lys211 that forms a putative phosphate binding site. Considering that RING0 is unique to parkin and that its hydrophobic interface with RING1 buries Cys431 in inactive parkin, targeting of phosphorylated Ub and/or Ubl towards this binding niche might be critical in dismantling autoinhibitory complexes during parkin activation. Function Mitophagy Parkin plays a crucial role in mitophagy and clearance of reactive oxygen species. Mitophagy is the elimination of damaged mitochondria in autophagosomes, and is dependent on a positive feedback cycle involving synergistic action of parkin and PINK1. Following severe cellular insult, rundown of mitochondrial membrane potential prevents import of PINK1 into the mitochondrial matrix and causes it to aggregate on the outer mitochondrial membrane (OMM). Parkin is recruited to mitochondria following depolarisation and phosphorylated by PINK1, which simultaneously phosphorylates Ub pre-conjugated to mitochondrial membrane proteins. PINK1 and Ub phosphorylation facilitate parkin activation and further assembly of mono- and poly-Ub chains. Considering the proximity of these chains to PINK1, further phosphorylation of Ub at Ser65 is likely, potentiating parkin mobilisation and substrate ubiquitination in a self-reinforcing cycle. Parkin substrates include mitofusins Mfn1 and