Structure of the second phosphoubiquitin-binding site in parkin

Abstract

Parkin and PINK1 regulate a mitochondrial quality control system that is mutated in some early onset forms of Parkinson's disease. Parkin is an E3 ubiquitin ligase and regulated by the mitochondrial kinase PINK1 via a two-step cascade. PINK1 first phosphorylates ubiquitin, which binds a recruitment site on parkin to localize parkin to damaged mitochondria. In the second step, PINK1 phosphorylates parkin on its ubiquitin-like domain (Ubl), which binds a regulatory site to release ubiquitin ligase activity. Recently, an alternative feed-forward mechanism was identified that bypasses the need for parkin phosphorylation through the binding of a second phosphoubiquitin (pUb) molecule. Here, we report the structure of parkin activated through this feed-forward mechanism. The crystal structure of parkin with pUb bound to both the recruitment and regulatory sites reveals the molecular basis for differences in specificity and affinity of the two sites. We use isothermal titration calorimetry measurements to reveal cooperativity between the two binding sites and the role of linker residues for pUbl binding to the regulatory site. The observation of flexibility in the process of parkin activation offers hope for the future design of small molecules for the treatment of Parkinson's disease.

Keywords: Parkinson disease; X-ray crystallography; analytical ultracentrifugation; feed-forward control; isothermal titration calorimetry; protein phosphorylation; protein structure; ubiquitin; ubiquitin ligase.