Abstract
Hereditary Parkinson’s disease is commonly caused by mutations in the protein kinase PINK1 or the E3 ubiquitin ligase Parkin, which function together to eliminate damaged mitochondria. PINK1 phosphorylates both Parkin and ubiquitin to stimulate ubiquitination of dozens of proteins on the surface of the outer mitochondrial membrane. However, the mechanisms by which Parkin recognizes specific proteins for modification remain largely unexplored. Here, we show that the C-terminal GTPase (cGTPase) of the Parkin primary substrate human Miro is necessary and sufficient for efficient ubiquitination. We present several new X-ray crystal structures of both human Miro1 and Miro2 that reveal substrate recognition and ubiquitin transfer to be specific to particular protein domains and lysine residues. We also provide evidence that Parkin substrate recognition is functionally separate from substrate modification. Finally, we show that prioritization for modification of a specific lysine sidechain of the cGTPase (K572) within human Miro1 is dependent on both its location and chemical microenvironment. Activation of Parkin by phosphorylation or by binding of pUb is required for prioritization of K572 for modification, suggesting that Parkin activation and acquisition of substrate specificity are coupled.
Highlights
Targeting, some E3 ligases exhibit low selectivity, modifying a ‘ubiquitination zone’ that is conformationally accessible to the catalytic machinery[27,30]
Using an in vitro reconstitution system consisting of purified human E1 (UBE1), E2 (UbcH7), and Ub, we found that both full-length 6xHis-tagged hMiro[1] and hMiro[2] are directly ubiquitinated by human Parkin phosphorylated at Serine 65 (S65) (p-S65 Parkin; Fig. 2a,b, Supplementary Fig. S3a,b)
Introduction of either decreased overall ubiquitination levels of hMiro1-CM; in contrast, mutation of the targeted lysine itself, K572R, abrogated ubiquitination at that residue but did not significantly change overall ubiquitination levels. These findings demonstrate that neighboring residues contribute to the targeting of particular substrate lysines for modification, and suggest that residues P553 and D568 in particular may be important for overall recognition of the hMiro[1] cGTPase by p-S65 Parkin
Summary
Targeting, some E3 ligases exhibit low selectivity, modifying a ‘ubiquitination zone’ that is conformationally accessible to the catalytic machinery[27,30]. In the absence of pUb, Parkin is generally unable to synthesize Ub chains, instead favoring direct modification and multi-monoubiquitination of its targets[22] This behavior suggests a mechanism of primary substrate selection independent of, or preceding, pUb chain formation. Guided by several structures of human Miro domains, we generated a minimal hMiro[1] cGTPase substrate that recapitulates Parkin specificity, but which is monoubiquitinated primarily at a single lysine. Targeting of this prioritized residue by Parkin was critically dependent on sidechain position and chemical microenvironment. Our results provide a framework for understanding a critical step following activation of the Parkin ubiquitination pathway - the recognition and primary ubiquitination of its substrates
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
