How phosphoubiquitin activates Parkin

RESEARCH HIGHLIGHT How phosphoubiquitin activates Parkin

A recent report, solving the structure of a Parkin-phosphoubiquitin complex, greatly advances the understanding of the Parkin activation mechanism.

Pink one, a mitochondrial protein kinase, and Parkin, a cytosolic E three ubiquitin ligase, mutated in hereditary forms of early onset Parkinson's disease, function together in mitochondrial maintenance. Pink one is imported into healthy mitochondria with normal membrane potential, and then cleaved by the PARL protease and eventually degraded in the proteasome. When mitochondria lose their membrane potential, Pink one remains exposed on the mitochondrial outer surface, recruiting Parkin and activating its E three activity to trigger extensive mitochondrial protein ubiquitination and subsequent clearance of the damaged mitochondria through autophagy.

Parkin belongs to the RBR, RING-in-Between-RING, E three ligase family, which has two tandem RING domains - the RING one domain binds ubiquitin-charged E two enzymes and transfers the ubiquitin to a catalytic cysteine residue in the RING two domain before conjugating it to a substrate. Parkin E three activity is autoinhibited through multiple intramolecular interactions: E two binding is blocked by the N-terminal ubiquitin-like domain as well as a 'repressor' element; the catalytic cysteine in the RING two domain is masked by a unique Parkin domain, also known as RING zero. Parkin E three activity is stimulated by Pink one through two Ser sixty-five phosphorylation events, one on the Parkin ubiquitin-like domain and the other on the equivalent serine in ubiquitin. Ubiquitin-like domain phosphorylation causes its dissociation from the RBR core; however, the mechanism through which phosphoubiquitin activates Parkin is unclear.

To understand how phosphoubiquitin induces Parkin activation, Wauer and colleagues obtained a crystal structure of truncated Parkin, one hundred forty to four hundred sixty-one, in complex with phospho-Ser sixty-five ubiquitin. They used human body louse Parkin, which contains a cysteine near its ubiquitin-binding site that can react with a ubiquitin suicide probe to form a stable complex for purification. The structure reveals that phosphoubiquitin has extensive interactions with multiple domains of Parkin. Phosphoubiquitin interacts with the RING one domain through its hydrophobic Ile forty-four patch, which is used for many Ub interactions; and its C-terminus also forms a close contact with the IBR domain.

The structure was further confirmed by a series of compelling functional analyses. Mutations in the predicted phosphate-binding pocket of Parkin, such as His three hundred two, greatly decreased phosphoubiquitin binding. Importantly, a Parkinson's disease patient mutation, was among them, thus supporting the physiological importance of phosphoubiquitin binding in Parkin function. Parkin E three activity can be activated by phosphoubiquitin in autoubiquitination assays even in the absence of ubiquitin-like domain phosphorylation. Several recent studies suggest that phosphoubiquitin binding is necessary for Parkin mitochondrial translocation. Consistently, these phosphoubiquitin binding mutations not only impaired the phosphoubiquitin-induced Parkin activation in autoubiquitination assays, but also crippled Parkin mitochondrial recruitment and localization.

Phosphoubiquitin binding induces profound structural changes that could explain Parkin activation. Phosphoubiquitin is bound to a straight helix in RING one; in contrast, this helix is kinked in the inactive Parkin. The straightening of this helix may trigger the rotation and movement of the IBR domain, thus stretching the IBR-repressor linker, which in turn affects the binding interface between RBR and the ubiquitin-like domain. Indeed, the authors showed that phosphoubiquitin can compete with and displace the ubiquitin-like domain from the Parkin RBR core. Moreover, phosphoubiquitin binding appears to stimulate Parkin Ser sixty-five phosphorylation by Pink one, as reported by Kazlauskaite et al.. Using nuclear magnetic resonance, Wauer and colleagues showed that ubiquitin-like domain phosphorylation disrupts its Ile forty-four patch, a binding interface between ubiquitin-like and RBR core, further ensuring the dissociation of ubiquitin-like domain and also explaining why phospho-Ser sixty-five ubiquitin-like cannot bind to the phospho-Ser sixty-five ubiquitin-binding pocket.

Do we completely understand Parkin activation? A puzzling observation is that ubiquitin-vinyl sulfone, which can conjugate to the catalytic cysteine when it is accessible, reacted with phospho-ubiquitin-like Parkin but not phosphoubiquitin-bound Parkin, indicating that the catalytic cysteine may not be completely exposed in the latter case. One might ask whether the autoubiquitination of phosphoubiquitin-activated Parkin occurs through the catalytic cysteine, or alternatively is due to RING one activity alone. Is ubiquitin-like phosphorylation essential for exposing the catalytic cysteine? Ubiquitin-like domain deletion greatly diminishes ubiquitin trapping by Parkin C four hundred thirty-one S during mitophagy, indicating a positive role of ubiquitin-like domain in Parkin activity. In the current work, the authors proposed an intriguing hypothesis: the phospho-ubiquitin-like domain may instead bind to the unique Parkin domain through a second, distinct putative phosphate-binding pocket. Consistent with this, they showed that mutations of this pocket blocked the E three activity of phospho-ubiquitin-like Parkin.

tial for exposing the catalytic cysteine? UBL domain deletion greatly diminish- es ubiquitin trapping by Parkin C431S during mitophagy, indicating a positive role of UBL domain in Parkin activity [9]. In the current work, the authors proposed an intriguing hypothesis: the phosphoUBL domain may instead bind to the UPD through a second, distinct putative phosphate-binding pocket [3]. Consistent with this, they showed that mutations of this pocket blocked the E3 activity of phospho-UBL Parkin.

Parkin is reportedly capable of catalyzing multiple different ubiquitin linkage chain types, including K six, K eleven, K forty-eight, and K sixty-three. Interestingly, in cells expressing only S sixty-five A ubiquitin, the abundance of K six, K eleven, and K forty-eight chains detected on mitochondria decrease dramatically. Could Parkin have multiple different activation states: ubiquitin-like phosphorylation alone, activation by phosphoubiquitin binding alone, or both? In this regard, it would be worth comparing the chain types formed by phospho-ubiquitin-like Parkin and phosphoubiquitin-activated Parkin. Another issue is which E two is used by Parkin during mitophagy. UBE two D and UBE two L three have been shown to redundantly activate Parkin at the initial stage and be required for Parkin mitochondrial translocation, whereas UBE two N appears to act at a later stage in mitochondrial clustering. It would be interesting to see whether Parkin associated with different E two enzymes would catalyze different chain linkage types.

Because of its clinical importance and clearly-defined function in mitophagy, a key cellular quality control process, Parkin's biochemical mechanism is being rapidly unfolded. This will not only provide therapeutic opportunities, such as rescue of some Parkin mutants, but also generate valuable insights into this newly-identified RING-HECT hybrid E three ligase family.