Abstract
Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.
Highlights
Parkinson’s disease is one of the most common neurodegenerative diseases
Using isothermal titration calorimetry (ITC), NMR, and small-angle X-ray scattering (SAXS), we show that phosphorylation of the ubiquitin-like domain (Ubl) domain releases its interaction with the Parkin RING1 domain and that this increases the affinity of pUb binding to Parkin
Parkin consists of an N-terminal Ubl domain, followed by a long linker leading to four zinc-binding domains: RING0, RING1, IBR, and RING2, collectively referred to as the R0RBR module
Summary
Parkinson’s disease is one of the most common neurodegenerative diseases. Its characteristic motor symptoms are caused by the loss of dopaminergic neurons in the substantia nigra area of the midbrain. Parkin ubiquitinates mitochondrial outer membrane proteins such as Mitofusin or Miro to induce a wide range of outcomes, from proteasomal degradation to vesicle formation, motility arrest, and mitochondrial autophagy (Narendra et al, 2008; Gegg et al, 2010; Tanaka et al, 2010; Ziviani et al, 2010; Wang et al, 2011; McLelland et al, 2014). All these Parkin-dependent quality control processes require PINK1 (PTEN-induced putative kinase 1), a Ser/Thr kinase with a mitochondrial targeting sequence. For example, upon depolarization, PINK1 accumulates on the cytosolic face of the outer membrane (Zhou et al, 2008) where it can recruit and activate Parkin (Geisler et al, 2010; Matsuda et al, 2010; Narendra et al, 2010)
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