Parkin Structure and Function
Parkin Structure and Function
Saturday, 14 February 2015
Exhibit Hall (San Jose Convention Center)
Mutations in the Parkin or PINK1 (PTEN-induced putative kinase protein 1) genes are the leading cause of autosomal recessive form of Parkinson’s disease (PD). The gene products parkin and PINK1 are neuroprotective proteins, which are involved in a common pathway regulating clearance of depolarized or damaged mitochondria by autophagy (termed mitophagy).Parkin is a 465-residue E3 ubiquitin ligase involved in ubiquitination pathway. Ubiquitination typically marks proteins for degradation through the covalent attachment of ubiquitin and ubiquitin chains to lysine residues in a substrate protein. In addition to degradation via the proteasome, ubiquitination can act as a signal for autophagy – degradation via lysosomes – as well as alter substrate protein activity or location. Parkin ubiquitinates mitochondrial proteins on the damaged organelle leading to their autophagic elimination. Ubiquitination is carried out through the sequential action of three enzymes: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. Parkin consists of a ubiquitin-like domain (Ubl) at its N-terminus and four zinc-coordinating RING-like domains: RING0, RING1, In-Between-RING (IBR), and RING2. It is believed that parkin may function as a RING/HECT hybrid, where ubiquitin is first transferred by the E2 enzyme onto parkin active cysteine and then to the substrate. Compared to other ubiquitin ligases, the Parkin protein exhibits low basal activity and requires activation both in vitro and in cells. Recently, we reported the crystal structure of full-length parkin using X-ray crystallography. This structure shows parkin in a compact auto-inhibited conformation and provides insight into how it is activated. In the structure RING0 occludes the ubiquitin acceptor site Cys431 in RING2 whereas a novel repressor element of parkin (REP) binds RING1 and blocks its E2-binding site. The ubiquitin-like domain (Ubl) binds adjacent to the REP through the hydrophobic surface centered around Ile44 and regulate parkin activity. Mutagenesis and NMR titrations verified interactions observed in the crystal. We also proposed the putative E2 binding site on RING1 and confirmed it by mutagenesis and NMR titrations. In mitophagy pathway, PINK1 acts upstream of parkin and is required for both parkin activation and recruitment to depolarized mitochondria. In this pathway, PINK1 phosphorylates parkin Ubl domain and ubiquitin both regulating Parkin ligase activity. In cells, expression of the non-phosphorylatable S65A ubiquitin delays parkin recruitment to the depolarized mitochondria, and mutation of both parkin and ubiquitin at Ser65 entirely abolishes parkin activation. Current work is directed towards obtaining structure of parkin in the active conformation in complex with E2 and phospho-ubiquitin. It is my hope that understanding how parkin functions will lead to new therapeutic strategies for treating and ultimately preventing PD.