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Parkinson’s: Completing the Parkin activation puzzle

Related publication: Gladkova C., Maslen S. L., Skehel J. M. & Komander D. Mechanism of parkin activation by PINK1. Nature 559, 410–414 (2018). DOI: 10.1038/s41586-018-0224-x

Publication keywords: Ubiquitin; Parkinson’s disease; Parkin; E3 ligase; PINK1; Mitophagy

Although Parkinson’s disease is the second most common neurodegenerative disorder, current diagnosis is subjective and there is no cure. Studying inherited, young-onset forms of the disease has helped to identify defective cellular processes that give rise to disease symptoms. Understanding these could help design more effective diagnostic tools or therapeutic strategies.

Parkin is an enzyme commonly defective in inherited forms of Parkinson’s disease. To maintain cellular health, Parkin attaches a small protein to damaged mitochondria, marking them for degradation. Although stabilising the active form of Parkin could form the basis of therapy, this state has evaded structural characterisation. Researchers therefore set out to capture the structure of active Parkin, using the Microfocus Macromolecular Crystallography (MX) beamline (I24), as their best crystals only reached 40 μm in two of the dimensions.

They found that dramatic structural rearrangements occur upon Parkin activation on damaged mitochondria, and identified a novel element essential for full Parkin activation. Being able to compare the active Parkin structure with previously resolved inactive forms is the first step to designing novel Parkinson’s disease therapies.

Although the work described here provides the final piece of the Parkin activation conundrum, Diamond Light Source has been instrumental in understanding the whole mechanism. This research group and others have previously resolved inactive Parkin states on beamlines I04 and I02 (highlighted in the ‘Insights into Parkinson’s disease from crystal structures of Parkin’ article in the Diamond Light Source Annual Review 2015/16).

: Figure 1: Completing the structural picture of Parkin activation. a) Autoinhibited Parkin, PDB ID: 5C1Z3. The surface of core Parkin domains (blue) is shown. The positions of the Ubl (green), REP (red)
and RING2 (cyan) domains disable Ub transfer; b) Autoinhibited Parkin bound to phospho-Ub (orange), PDB ID: 5N2W<sup>5</sup>. Although core Parkin domains rearrange to bind phospho-Ub, autoinhibitory
elements persist; c) Active phospho-Parkin bound to phospho-Ub, PDB ID: 6GLC. The phospho-Ubl and ACT (light green) displace the catalytic RING2 domain, thus activating Parkin; d) An overlay
between b) and c) showing surface of the phospho-Ubl and RING2 domains, coloured as above.

Parkinson’s Disease (PD) is the second most common neurodegenerative disorder; it is characterised by neuronal loss and a subsequent decrease of dopamine levels in the brain. While most cases of PD arise sporadically, 10% of patients carry mutations giving rise to a young-onset inherited form of the disease. Among genes mutated in young-onset PD are the kinase PINK1 and the E3 ubiquitin ligase Parkin, an enzyme which attaches the small signalling protein ubiquitin (Ub) to substrates. In cells PINK1 and Parkin orchestrate clearance of damaged mitochondria by a specialised form of autophagy, known as mitophagy¹.

Under resting conditions, PINK1 is continuously turned-over, while Parkin resides in the cytosol. Previous structural work by others and us has explained how the activity of cytosolic Parkin is restrained by several autoinhibitory elements. To induce mitophagy, PINK1 is stabilised on the outer mitochondrial membrane of damaged mitochondria and phosphorylates Ub as its first substrate. Mitochondrially anchored phosphorylated Ub (phospho-Ub) binds to Parkin and triggers Parkin translocation to the damaged organelles. There, the N-terminal Ub-like (Ubl) domain of Parkin becomes a second substrate for PINK1. Only phosphorylated Parkin is fully active, although the nature of the Parkin rearrangement upon phosphorylation remained a key unanswered question in the field¹.

Before transferring Ub to mitochondrial substrates, Parkin receives an activated Ub-molecule onto its active site Cys residue from an upstream E2 Ub-conjugating enzyme. However, in cytosolic Parkin the conserved binding site for the E2 enzyme is blocked and the catalytic Cys is buried in a hydrophobic interface between the catalytic RING2 domain and the unique Parkin domain (UPD), (Fig. 1a). To enable E2 binding the Repressor Element of Parkin (REP) and the N-terminal Ubl domain must be displaced from their autoinhibitory binding sites. Furthermore, release of the catalytic Cys residue requires a large-scale rearrangement where the catalytic RING2 domain is entirely displaced from its autoinhibitory binding site on the UPD^2,3.

Our group has previously reported a structure of Parkin bound to phospho- Ub4. While phospho-Ub binding mediates Parkin translocation to mitochondria and primes Parkin for phosphorylation, most autoinhibitory elements remain intact (Fig. 1b). Helix straightening necessary to create an optimised phospho-Ub binding site weakens the autoinhibitory Ubl interface. The Ubl is therefore released upon phospho-Ub binding and becomes accessible for phosphorylation. Despite these rearrangements, the resolved complex of Parkin bound to phospho-Ub remains inactive, as the catalytic Cys residue remains buried and the REP remains in place^4,5.

: Figure 2: Activating Parkin. a) The ACT linchpin (light green) stabilises the new activating
Parkin interface by contacting both the UPD (dark blue) and the phospho-Ubl domain
(green); b) Overlay with autoinhibited Parkin (PDB ID: 5N2W5) shows how the ACT mimics
autoinhibitory interactions between the catalytic RING2 domain (cyan) and the UPD domain
to activate Parkin; c) A schematic representation of the Parkin activation cascade, domains
coloured as above.

Although previous Parkin structures do not capture the active state of the enzyme, they provide a structural basis for many disease-linked mutations. Intriguingly, some mutations could not be explained to date as they likely disrupt the unknown active state. Among these are mutations of a phosphate-binding pocket previously identified on the UPD as well as mutations in a linker region between the Ubl domain and the rest of Parkin. Although partially conserved, this linker region had previously been omitted from analysis due to its intrinsic flexibility.

Dynamic and proteolytic studies suggested that upon activation, the catalytic RING2 domain is released and the catalytic Cys becomes accessible, consistent with a fully active state of Parkin. To overcome the difficulties of crystallising full-length activated phospho-Parkin with a flexible RING2 domain, a cleavage site within the protein was designed so that the flexibly linked RING2 can be removed following activation by PINK1 phosphorylation in vitro.

The 1.8 Å structure of human phospho-Parkin(ΔRING2), determined from data collected at the Microfocus MX beamline, finally revealed the extensive domain rearrangements required to release the catalytic domain from its autoinhibitory position and enable Parkin activity (Fig. 1c). While the core domains remained unperturbed upon phosphorylation, the phospho-Ubl domain shifted by over 50 Å compared to previous structures. In the activated form of the protein, the phospho-Ubl binds to a new activating interface, which partially overlaps with the previously observed RING2 autoinhibitory binding site (Fig. 1d). Simultaneous binding of both domains is sterically impossible. In a carefully balanced system, intramolecular binding of the phospho-Ubl, therefore, displaces the catalytic RING2 domain to activate phospho-Parkin. The phosphorylated residue in the Ubl is placed in the mutation-lined phosphate pocket on the UPD, elucidating the basis of active-state specific pathogenic Parkin mutations.

Most strikingly, a conserved portion of the linker region which follows the N-terminal Ubl domain could be resolved. Since mutation or deletion of the region led to severely diminished Parkin activity, we termed the conserved region the Activating Element of Parkin (ACT). The ACT binding mode mimics the interactions which keep the RING2 domain in its autoinhibitory position (Fig. 2a,b). The amphipathic nature of the ACT allows insertion of hydrophobic residues into the groove of the UPD, while polar residues contact the phospho- Ubl domain, stabilising the novel activating interface. Interestingly, Arg104, which electrostatically interacts with the phospho-Ubl, is mutated in patients suffering from young-onset PD.

Our structure contributes the last missing piece of the Parkin activation cascade. Only now can we fully appreciate the scale of domain rearrangements necessary to activate Parkin specifically on the surface of damaged mitochondria (Fig. 2c). Initially, Parkin adopts an almost serpentine arrangement, where regions linking Ubl and catalytic RING2 domains to the Parkin core cross over to enable the domains to adopt their autoinhibitory positions. Upon phospho-Ub binding, the Ubl domain is released for phosphorylation by PINK1, although the catalytic RING2 domain remains sequestered. Upon phosphorylation the phospho-Ubl domain together with the ACT element liberate the catalytic RING2 domain, enabling Ub transfer activity and completely unravelling Parkin.

Now that the relevant Parkin states have been structurally characterised, it is clear that structural facets unique to the active state exist and could be exploited translationally. Modulating Parkin conformations by designing conformation-specific molecules might benefit PD sufferers in the future.

References:

Harper J. W. et al. Building and decoding ubiquitin chains for mitophagy. Nat Rev Mol Cell Biol. 19, 93–108 (2018). DOI: 10.1038/nrm.2017.129
Wauer T. et al. Structure of the human Parkin ligase domain in an autoinhibited state. EMBO J. 32, 2099–2112 (2013). DOI: 10.1038/ emboj.2013.125
Kumar A. et al. Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis. EMBO J. 34, 2506–2521 (2015). DOI: 10.15252/embj.201592337
Wauer T. et al. Mechanism of phospho-ubiquitin-induced PARKIN activation. Nature 524, 370-374 (2015). DOI: 10.1038/nature14879
Kumar A. et al. Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity. Nat. Struct. Mol. Biol. 24, 475–483 (2017). DOI: 10.1038/nsmb.3400

Funding acknowledgement:

Access to Diamond Light Source was supported in part by the EU FP7 infrastructure grant BIOSTRUCT-X (contract no. 283570).

Work at the Komander laboratory was supported by the Medical Research Council (U105192732), the European Research Council (724804), the Michael J. Fox Foundation and the Lister Institute for Preventive Medicine.

Corresponding authors:

Christina Gladkova, MRC Laboratory of Molecular Biology, [email protected] and Dr David Komander, Walter and Eliza Hall Institute of Medical Research, [email protected]

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Parkinson’s: Completing the Parkin activation puzzle