Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra leading to motor defects. PD is primarily sporadic, occurring mainly in older people. Mutations in several genes, such as PARK2 (Parkin) and PARK6 (PINK1, PTEN-induced kinase 1), cause early-onset autosomal recessive juvenile parkinsonism (ARJP). Parkin and PINK1 function together in a common mitochondrial homeostasis pathway in which damaged mitochondria are cleared by autophagy (mitophagy; Bonifati et al., 2002; Martin et al., 2011; Kitada et al., 1998; Valente et al., 2004; Chung et al., 2016; Exner et al., 2012; Narendra et al., 2012).

Parkin is an autoinhibited RBR family E3 ubiquitin ligase (Chaugule et al., 2011) consisting of an N-terminal ubiquitin-like (Ubl) domain followed by four Zn²⁺ binding domains RING0, RING1, in-between-RING (IBR), and RING2 (Spratt et al., 2014). Parkin is a cytosolic protein activated following mitochondrial stress, mediated by PINK1 phosphorylation of Serine 65 (S65) on ubiquitin. Phosphorylation of ubiquitin enhances binding with Parkin and leads to the recruitment of Parkin to sites of damaged mitochondria (Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014). On mitochondria, S65 of the Ubl domain of Parkin is phosphorylated by PINK1 (Kazlauskaite et al., 2014; Kondapalli et al., 2012; Shiba-Fukushima et al., 2014; Shiba-Fukushima et al., 2012), resulting in a fully active Parkin conformation. Fully active Parkin attaches new ubiquitin molecules on mitochondrial proteins, which are phosphorylated by PINK1 to recruit more cytoplasmic Parkin to the mitochondria, thus resulting in a positive feedforward amplification cycle (Ordureau et al., 2014). Ubiquitination of mitochondrial proteins by Parkin also leads to the recruitment of autophagy receptors required for mitophagy (Tanaka et al., 2010; Chan et al., 2011).

Like other RBR-family E3 ligases, Parkin binds to an E2, and ubiquitin is transferred from E2 onto the catalytic C431 residue (on RING2) of Parkin before ubiquitination of lysines on target substrates (Wenzel et al., 2011; Walden and Rittinger, 2018). On Parkin, several elements are present that maintain autoinhibited conformation of Parkin. The E2 binding site on RING1 is blocked by the Ubl domain and the short repressor (REP) element. Furthermore, C431 on RING2 is occluded by the RING0 domain of Parkin, which inhibits Parkin activity (Chaugule et al., 2011; Riley et al., 2013; Trempe et al., 2013; Wauer et al., 2015). The phospho-Ubl domain binds within a basic patch (comprising K161, R163, and K211) on RING0 and displaces RING2 to expose C431 to activate Parkin (Gladkova et al., 2018). In the structure of phospho-Parkin with RING2 removed, an activating element (ACT, 101–109), which is present in the linker region (77-140) between Ubl and RING0 domains, binds on the RING0 interface (Gladkova et al., 2018). Mutations in the ACT are shown to affect Parkin activity negatively (Gladkova et al., 2018), suggesting their importance in Parkin regulation. Phospho-ubiquitin (pUb) binds in a pocket between RING0 and RING1, and activates Parkin allosterically (Wauer et al., 2015; Sauvé et al., 2015; Kumar et al., 2015). pUb binding results in the displacement of the IBR domain, and the straightening of helix-1 of the RING1 domain (Kumar et al., 2017). Massive domain rearrangements have been proposed in the active state to allow the transfer of donor ubiquitin (bound between helix-1 and IBR) from E2 (on RING1) to C431 (on RING2) of Parkin (Gladkova et al., 2018; Kumar et al., 2017; Sauvé et al., 2018; Condos et al., 2018).

Several crystal structures of Parkin were solved in the last decade using various truncations in Parkin, which revealed new insights into the conformational changes during the intricate activation process of Parkin (Figure 1—figure supplement 1A). A few years ago, using the structure of truncated phospho-Parkin (RING2 removed; Figure 1—figure supplement 1A), a model of phospho-Parkin was proposed wherein RING2 would be displaced from RING0 to occupy a pocket near the IBR domain (Figure 1—figure supplement 1B; Gladkova et al., 2018; Sauvé et al., 2018). However, the extent of conformational changes and domain rearrangements due to different regulatory elements of Parkin in the active state remains elusive. For example, it is not clear how and by what mechanism the displaced pUbl from RING1 would be recognized on RING0 in the cis molecule (as per the proposed model in Figure 1—figure supplement 1B) and not in the trans molecule, especially considering the likelihood of an encounter with a trans molecule in the crowded molecular environment. Previous cellular data co-expressing WT-Parkin and mutant Parkin constructs suggested the self-association of Parkin molecules after PINK1 activation at sites of damaged mitochondria (Lazarou et al., 2013). However, a role for phospho-ubiquitin-mediated recruitment of mutant Parkin, induced by co-expressed wild-type Parkin, could not be excluded. Furthermore, structural studies to understand the Parkin activation mechanism in the last decade have not captured any dimerization of Parkin in vitro (Wenzel et al., 2011; Walden and Rittinger, 2018; Riley et al., 2013; Trempe et al., 2013; Wauer and Komander, 2013; Gladkova et al., 2018; Wauer et al., 2015; Kumar et al., 2015; Sauvé et al., 2015; Kumar et al., 2017; Sauvé et al., 2018; Condos et al., 2018).

Herein, using X-ray crystal structures, biophysical methods, and in vitro assays, we demonstrate the trans conformational changes in Parkin during the activation process, revealing novel insights into the Parkin activation mechanism. Our data suggest that the phospho-Ubl (pUbl) domain transiently binds to the basic patch on RING0 and competes with the RING2 domain. In addition to the previous observation that pUbl binding results in RING2 displacement, our new data show that the presence of RING2 restricts the binding of pUbl with the Parkin core, which establishes the competitive mode of interaction between RING2 and pUbl. The crystal structure of pUbl-linker (1-140) depleted Parkin (141-465)-pUb complex and supporting data show that RING2 is displaced transiently during the activation process and returns to its closed state after the removal of the pUbl domain from phospho-Parkin, suggesting dynamic nature of conformational changes during Parkin activation. Furthermore, we report Parkin dimerization, mediated by interactions between pUbl and the basic patch on the RING0 domain in trans. We also demonstrate that phospho-Parkin activates autoinhibited Parkin in trans, suggesting an additional feedforward mechanism of Parkin activation. Our data also reveals new insights into the regulation mediated by the ACT of Parkin, wherein the ACT is required for maintaining the enzyme kinetics. We show that similar to phospho-Ubl, ACT can also work in trans, although ACT is more efficient in cis. Furthermore, using X-ray crystallography and supporting experiments, we have characterized a new donor ubiquitin binding site in the linker region (408-415) between the REP element and RING2, which plays a crucial role in Parkin activity.

Autoinhibition of Parkin is mediated by several mechanisms. Ubl domain and REP element block the E2 binding site on RING1 (Chaugule et al., 2011; Trempe et al., 2013; Kumar et al., 2015; Sauvé et al., 2015), whereas the RING0 domain occludes the catalytic C431 on RING2. A few years after the discovery of Parkin autoinhibition, various groups discovered PINK1-mediated phosphorylation of S65 on the ubiquitin and Ubl domain of Parkin, leading to the activation of Parkin (Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Kondapalli et al., 2012). In the last few years, several structural studies have aimed to understand the conformational changes in Parkin that are driven by phosphorylation leading to Parkin activation. The structure of RING2 truncated phospho-Parkin (1-382) in complex with pUb showed the pUbl domain of Parkin bound to the basic patch (comprising K161, R163, K211) on RING0, which led to the displacement of RING2 and REP during Parkin activation (Gladkova et al., 2018; Sauvé et al., 2018). Previous studies using various biophysical methods reported a K_d of ~2 µM between Ubl and R0RBR/∆Ubl-Parkin; however, pUbl showed no interaction, which led to the proposed mechanism suggesting displacement of the pUbl domain to activate Parkin (Kumar et al., 2015; Sauvé et al., 2015).

Our data show that RING2 and pUbl compete for binding on the basic patch of RING0 (Figure 2). Our data also show that RING2 and REP displacement after Parkin phosphorylation is transient; RING2 and REP return to their original position after removal of the pUbl from phospho-Parkin (Figure 2). Our data explains that due to the net high concentration of the fused domain (RING2 or pUbl), and competitive mode of interaction, binding/displacement of pUbl/RING2 domain in trans couldn’t be observed in the previous studies. However, untethering of pUbl/RING2 overcomes the latter issue, and trans interaction between Parkin molecules can be observed. By untethering the linker between RING2 and IBR, after pUbl binding, the displaced RING2 is no longer able to return to the RING0 pocket, thus the binding of pUbl on the basic patch of RING0 is stabilized (Figure 2). Untethered RING2 leads to a strong affinity between phospho-Ubl and core of Parkin with K_d around 1 µM (Figures 4 and 5), which is also supported by complex formation on SEC/SEC-MALS using phospho-Parkin and Parkin (Figure 5).

A feedforward control mechanism was suggested in the PINK1-Parkin pathway wherein PINK1-dependent phosphorylation of ubiquitin and Parkin leads to Parkin activation on mitochondria (Kazlauskaite et al., 2014; Shiba-Fukushima et al., 2012; Ordureau et al., 2014; Zhuang et al., 2016; Tang et al., 2017). However, biophysical studies aimed to understand Parkin activity did not show any dimerization of Parkin or Parkin-Parkin association in trans (Wenzel et al., 2011; Walden and Rittinger, 2018; Riley et al., 2013; Trempe et al., 2013; Wauer et al., 2015; Kumar et al., 2017; Sauvé et al., 2018; Condos et al., 2018; Wauer and Komander, 2013; Gladkova et al., 2018; Kumar et al., 2015; Sauvé et al., 2015). Our data demonstrate that phospho-Parkin and WT-Parkin can form a stable complex in trans to mediate Parkin dimerization (Figure 5). We also show that phospho-Parkin can activate WT-Parkin in trans, reaffirming that a major mode of Parkin autoinhibition is mediated by RING0 blocking the catalytic C431 on the RING2 domain. Furthermore, our data suggest an additional feedforward activation model of Parkin wherein fully-activated Parkin (phospho-Parkin bound to pUb) molecules can activate partially-activated Parkin (WT-Parkin bound to pUb) molecules, which is mediated by interactions between pUbl and RING0 in trans (Figure 10). The latter can be relevant in the context of healthy carriers of heterozygous mutations on Parkin. The critical role of pUbl supports data showing the importance of Ubl phosphorylation in vivo, as demonstrated by the discovery of Parkinson’s patients associated with homozygous S65N Parkin mutation (McWilliams et al., 2018). This data also highlights the importance of various Parkin isoforms that have been identified (Figure 5—figure supplement 1), especially the ones that lack Ubl domain or REP-RING2 domains, as they can complement each other using our proposed trans model in Figure 10.

Figure 10

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ACT was proposed to have a role in Parkin activation, as it was shown that the deletion/mutation of ACT leads to the loss of Parkin activity (Gladkova et al., 2018). We demonstrate that ACT plays a key role due to its inherent capacity to bind with the RING0 pocket. Unlike other functional mutations on Parkin affecting interaction with E2 or Ub_don, ACT deletion does not affect binding with E2~Ub_don (Figure 7). We show that ACT plays a crucial role in enzyme kinetics and only slows the Parkin activity, possibly by affecting the inherently dynamic nature of RING2 (Figure 7). Furthermore, we also demonstrate that although ACT can be complemented in trans, ACT on a cis molecule is more effective (Figure 8).

The linker connecting IBR and RING2 of Parkin comprises two components: a REP element (391-405) and a flexible linker (408-415). Various Parkin structures solved so far show REP element blocking the E2 binding site on RING1; however, linker (408-415) remained flexible in most structures, and its role remained elusive. Interestingly, pathogenic mutation T415N in the linker region was shown to abolish the E3 ligase activity of Parkin (Chaugule et al., 2011). Also, using peptide array analysis, Chaugule and colleagues proposed a Parkin Ubl/ubiquitin-binding (PUB) site in the C-terminal domain of Parkin (Chaugule et al., 2011). Here, we demonstrate that the linker (408-415) interacts with donor ubiquitin (Ub_don) of E2~Ub_don (Figure 9). Although the linker between IBR-RING2 is not conserved across RBR family E3-ligases, the core of interactions between the linker and Ub_don is mediated by hydrophobic residue in the linker region (Figure 9). In the autoinhibited closed state of Parkin, the linker between IBR-RING2 of Parkin is present in a straight conformation, leading to IBR and RING2 occupying diagonally opposite conformation, which is quite similar to what is seen in HOIP RBR and E2~Ub_don complex structure (Figure 9, Figure 9—figure supplement 2A; Lechtenberg et al., 2016). However, the recent structures of RBR family E3-ligases (HHARI, RNF216, HOIL-1) (Horn-Ghetko et al., 2021; Wang et al., 2023) show a kinked conformation of the linker connecting IBR-RING2 (Figure 9—figure supplement 2A). Interestingly, the kink in the linker region plays a crucial role in bringing RING2 to the catalytically feasible state (Figure 9—figure supplement 2A). Conversely, under the extended conformation of the linker, catalytic feasibility is not possible (Figure 9—figure supplement 2A). The conformational flexibility in the linker (408-415) region of Parkin is also supported by the fact that it is disordered in most Parkin structures, or seen as a loop in a couple of Parkin structures, whereas AlphaFold predicts it as a helix similar to other RBR structures (Figure 9, Figure 9—figure supplement 1). Previous data observed the opening of RING2 after the addition of E2~Ub_don in R0RBR (Condos et al., 2018). The latter observation also suggests that conformational changes might be induced in the linker region after binding with donor ubiquitin or due to the movement of RING2, and needs further investigation. Also, as mentioned above, the conformation of donor ubiquitin and linker captured in the present study might be one of the possible intermediates. Although the regulatory mechanisms vary across RBR family E3-ligases, the catalytic core (IBR-RING2) undergoes similar conformational changes, leading to a unified catalysis mechanism in various RBR family E3-ligases.

Overall, our new structural and biophysical analysis elaborates a new understanding of Parkin activation and regulation that will aid in efforts to develop small molecular activators of Parkin as a therapeutic strategy for PD.

Structure coordinates were deposited in the protein data bank, and accession codes are included in Table 1. All data generated or analyzed in this study are included in the manuscript and supporting files; a manuscript source data file has been provided for all the figures.

1. 1. Lenka et al.

   (2024) RCSB Protein Data Bank

   ID 8IKM. Crystal Structure.

   https://www.rcsb.org/search?request=8IKM
2. 1. Lenka DR
   2. Kumar A

   (2024) RCSB Protein Data Bank

   ID 8IK6. Crystal Structure.

   https://www.rcsb.org/search?request=8IK6
3. 1. Lenka DR
   2. Kumar A

   (2024) RCSB Protein Data Bank

   ID 8JWV. Crystal Structure.

   https://www.rcsb.org/search?request=8JWV
4. 1. Lenka DR
   2. Kumar A

   (2024) RCSB Protein Data Bank

   ID 8IKT. Crystal Structure.

   https://www.rcsb.org/search?request=8IKT
5. 1. Lenka DR
   2. Kumar A

   (2024) RCSB Protein Data Bank

   ID 8IKV. Crystal Structure.

   https://www.rcsb.org/search?request=8IKV

1. 1. Berndsen CE
   2. Wolberger C

   (2011) A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins

   Analytical Biochemistry 418:102–110.

   https://doi.org/10.1016/j.ab.2011.06.034

   • PubMed
   • Google Scholar
2. 1. Bonifati V
   2. Dekker MCJ
   3. Vanacore N
   4. Fabbrini G
   5. Squitieri F
   6. Marconi R
   7. Antonini A
   8. Brustenghi P
   9. Dalla Libera A
   10. De Mari M
   11. Stocchi F
   12. Montagna P
   13. Gallai V
   14. Rizzu P
   15. van Swieten JC
   16. Oostra B
   17. van Duijn CM
   18. Meco G
   19. Heutink P
   20. Italian Parkinson Genetics Network

   (2002) Autosomal recessive early onset parkinsonism is linked to three loci: PARK2, PARK6, and PARK7

   Neurological Sciences 23 Suppl 2:S59–S60.

   https://doi.org/10.1007/s100720200069

   • PubMed
   • Google Scholar
3. 1. Borodovsky A
   2. Ovaa H
   3. Kolli N
   4. Gan-Erdene T
   5. Wilkinson KD
   6. Ploegh HL
   7. Kessler BM

   (2002) Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family

   Chemistry & Biology 9:1149–1159.

   https://doi.org/10.1016/s1074-5521(02)00248-x

   • PubMed
   • Google Scholar
4. 1. Chan NC
   2. Salazar AM
   3. Pham AH
   4. Sweredoski MJ
   5. Kolawa NJ
   6. Graham RLJ
   7. Hess S
   8. Chan DC

   (2011) Broad activation of the ubiquitin-proteasome system by parkin is critical for mitophagy

   Human Molecular Genetics 20:1726–1737.

   https://doi.org/10.1093/hmg/ddr048

   • PubMed
   • Google Scholar
5. 1. Chaugule VK
   2. Burchell L
   3. Barber KR
   4. Sidhu A
   5. Leslie SJ
   6. Shaw GS
   7. Walden H

   (2011) Autoregulation of parkin activity through its ubiquitin-like domain

   The EMBO Journal 30:2853–2867.

   https://doi.org/10.1038/emboj.2011.204

   • PubMed
   • Google Scholar
6. 1. Chung SY
   2. Kishinevsky S
   3. Mazzulli JR
   4. Graziotto J
   5. Mrejeru A
   6. Mosharov EV
   7. Puspita L
   8. Valiulahi P
   9. Sulzer D
   10. Milner TA
   11. Taldone T
   12. Krainc D
   13. Studer L
   14. Shim JW

   (2016) Parkin and pink1 patient ipsc-derived midbrain dopamine neurons exhibit mitochondrial dysfunction and α-synuclein accumulation

   Stem Cell Reports 7:664–677.

   https://doi.org/10.1016/j.stemcr.2016.08.012

   • PubMed
   • Google Scholar
7. 1. Collaborative Computational Project, Number 4

   (1994) The CCP4 suite: programs for protein crystallography

   Acta Crystallographica Section D Biological Crystallography 50:760–763.

   https://doi.org/10.1107/S0907444994003112

   • Google Scholar
8. 1. Condos TE
   2. Dunkerley KM
   3. Freeman EA
   4. Barber KR
   5. Aguirre JD
   6. Chaugule VK
   7. Xiao Y
   8. Konermann L
   9. Walden H
   10. Shaw GS

   (2018) Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

   The EMBO Journal 37:e100014.

   https://doi.org/10.15252/embj.2018100014

   • PubMed
   • Google Scholar
9. 1. Emsley P
   2. Cowtan K

   (2004) Coot: model-building tools for molecular graphics

   Acta Crystallographica. Section D, Biological Crystallography 60:2126–2132.

   https://doi.org/10.1107/S0907444904019158

   • PubMed
   • Google Scholar
10. 1. Exner N
    2. Lutz AK
    3. Haass C
    4. Winklhofer KF

    (2012) Mitochondrial dysfunction in parkinson’s disease: molecular mechanisms and pathophysiological consequences

    The EMBO Journal 31:3038–3062.

    https://doi.org/10.1038/emboj.2012.170

    • PubMed
    • Google Scholar
11. 1. Gladkova C
    2. Maslen SL
    3. Skehel JM
    4. Komander D

    (2018) Mechanism of parkin activation by PINK1

    Nature 559:410–414.

    https://doi.org/10.1038/s41586-018-0224-x

    • PubMed
    • Google Scholar
12. 1. Horn-Ghetko D
    2. Krist DT
    3. Prabu JR
    4. Baek K
    5. Mulder MPC
    6. Klügel M
    7. Scott DC
    8. Ovaa H
    9. Kleiger G
    10. Schulman BA

    (2021) Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly

    Nature 590:671–676.

    https://doi.org/10.1038/s41586-021-03197-9

    • PubMed
    • Google Scholar
13. 1. Kabsch W

    (2010) Acta Crystallogr

    Sect. D Biol. Crystallogr 66:125.

    https://doi.org/10.1107/S0907444909047337

    • Google Scholar
14. 1. Kane LA
    2. Lazarou M
    3. Fogel AI
    4. Li Y
    5. Yamano K
    6. Sarraf SA
    7. Banerjee S
    8. Youle RJ

    (2014) PINK1 phosphorylates ubiquitin to activate parkin E3 ubiquitin ligase activity

    The Journal of Cell Biology 205:143–153.

    https://doi.org/10.1083/jcb.201402104

    • PubMed
    • Google Scholar
15. 1. Kazlauskaite A
    2. Kondapalli C
    3. Gourlay R
    4. Campbell DG
    5. Ritorto MS
    6. Hofmann K
    7. Alessi DR
    8. Knebel A
    9. Trost M
    10. Muqit MMK

    (2014) Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

    The Biochemical Journal 460:127–139.

    https://doi.org/10.1042/BJ20140334

    • PubMed
    • Google Scholar
16. 1. Kazlauskaite A
    2. Martínez-Torres RJ
    3. Wilkie S
    4. Kumar A
    5. Peltier J
    6. Gonzalez A
    7. Johnson C
    8. Zhang J
    9. Hope AG
    10. Peggie M
    11. Trost M
    12. van Aalten DMF
    13. Alessi DR
    14. Prescott AR
    15. Knebel A
    16. Walden H
    17. Muqit MMK

    (2015) Binding to serine 65-phosphorylated ubiquitin primes parkin for optimal PINK1-dependent phosphorylation and activation

    EMBO Reports 16:939–954.

    https://doi.org/10.15252/embr.201540352

    • PubMed
    • Google Scholar
17. 1. Kitada T
    2. Asakawa S
    3. Hattori N
    4. Matsumine H
    5. Yamamura Y
    6. Minoshima S
    7. Yokochi M
    8. Mizuno Y
    9. Shimizu N

    (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism

    Nature 392:605–608.

    https://doi.org/10.1038/33416

    • PubMed
    • Google Scholar
18. 1. Kondapalli C
    2. Kazlauskaite A
    3. Zhang N
    4. Woodroof HI
    5. Campbell DG
    6. Gourlay R
    7. Burchell L
    8. Walden H
    9. Macartney TJ
    10. Deak M
    11. Knebel A
    12. Alessi DR
    13. Muqit MMK

    (2012) PINK1 is activated by mitochondrial membrane potential depolarization and stimulates parkin E3 ligase activity by phosphorylating Serine 65

    Open Biology 2:120080.

    https://doi.org/10.1098/rsob.120080

    • PubMed
    • Google Scholar
19. 1. Koyano F
    2. Okatsu K
    3. Kosako H
    4. Tamura Y
    5. Go E
    6. Kimura M
    7. Kimura Y
    8. Tsuchiya H
    9. Yoshihara H
    10. Hirokawa T
    11. Endo T
    12. Fon EA
    13. Trempe JF
    14. Saeki Y
    15. Tanaka K
    16. Matsuda N

    (2014) Ubiquitin is phosphorylated by PINK1 to activate parkin

    Nature 510:162–166.

    https://doi.org/10.1038/nature13392

    • PubMed
    • Google Scholar
20. 1. Kumar A
    2. Aguirre JD
    3. Condos TEC
    4. Martinez-Torres RJ
    5. Chaugule VK
    6. Toth R
    7. Sundaramoorthy R
    8. Mercier P
    9. Knebel A
    10. Spratt DE
    11. Barber KR
    12. Shaw GS
    13. Walden H

    (2015) Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

    The EMBO Journal 34:2506–2521.

    https://doi.org/10.15252/embj.201592337

    • PubMed
    • Google Scholar
21. 1. Kumar A
    2. Chaugule VK
    3. Condos TEC
    4. Barber KR
    5. Johnson C
    6. Toth R
    7. Sundaramoorthy R
    8. Knebel A
    9. Shaw GS
    10. Walden H

    (2017) Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity

    Nature Structural & Molecular Biology 24:475–483.

    https://doi.org/10.1038/nsmb.3400

    • PubMed
    • Google Scholar
22. 1. La Cognata V
    2. Maugeri G
    3. D’Amico AG
    4. Saccone S
    5. Federico C
    6. Cavallaro S
    7. D’Agata V

    (2018) Differential expression of PARK2 splice isoforms in an in vitro model of dopaminergic-like neurons exposed to toxic insults mimicking parkinson’s disease

    Journal of Cellular Biochemistry 119:1062–1073.

    https://doi.org/10.1002/jcb.26274

    • PubMed
    • Google Scholar
23. 1. Lazarou M
    2. Narendra DP
    3. Jin SM
    4. Tekle E
    5. Banerjee S
    6. Youle RJ

    (2013) PINK1 drives parkin self-association and HECT-like E3 activity upstream of mitochondrial binding

    The Journal of Cell Biology 200:163–172.

    https://doi.org/10.1083/jcb.201210111

    • PubMed
    • Google Scholar
24. 1. Lechtenberg BC
    2. Rajput A
    3. Sanishvili R
    4. Dobaczewska MK
    5. Ware CF
    6. Mace PD
    7. Riedl SJ

    (2016) Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

    Nature 529:546–550.

    https://doi.org/10.1038/nature16511

    • PubMed
    • Google Scholar
25. 1. Lenka DR
    2. Chaurasiya S
    3. Kumar A

    (2023) Intricate mechanism (s) of substrate specificity and loss of function on disease mutation (k211n) of parkin

    Biochemistry 01:e8018.

    https://doi.org/10.1101/2023.11.21.568018

    • Google Scholar
26. 1. Martin I
    2. Dawson VL
    3. Dawson TM

    (2011) Recent advances in the genetics of parkinson’s disease

    Annual Review of Genomics and Human Genetics 12:301–325.

    https://doi.org/10.1146/annurev-genom-082410-101440

    • PubMed
    • Google Scholar
27. 1. McWilliams TG
    2. Barini E
    3. Pohjolan-Pirhonen R
    4. Brooks SP
    5. Singh F
    6. Burel S
    7. Balk K
    8. Kumar A
    9. Montava-Garriga L
    10. Prescott AR
    11. Hassoun SM
    12. Mouton-Liger F
    13. Ball G
    14. Hills R
    15. Knebel A
    16. Ulusoy A
    17. Di Monte DA
    18. Tamjar J
    19. Antico O
    20. Fears K
    21. Smith L
    22. Brambilla R
    23. Palin E
    24. Valori M
    25. Eerola-Rautio J
    26. Tienari P
    27. Corti O
    28. Dunnett SB
    29. Ganley IG
    30. Suomalainen A
    31. Muqit MMK

    (2018) Phosphorylation of parkin at serine 65 is essential for its activation in vivo

    Open Biology 8:180108.

    https://doi.org/10.1098/rsob.180108

    • PubMed
    • Google Scholar
28. 1. Mirdita M
    2. Schütze K
    3. Moriwaki Y
    4. Heo L
    5. Ovchinnikov S
    6. Steinegger M

    (2022) ColabFold: making protein folding accessible to all

    Nature Methods 19:679–682.

    https://doi.org/10.1038/s41592-022-01488-1

    • PubMed
    • Google Scholar
29. 1. Murshudov GN
    2. Skubák P
    3. Lebedev AA
    4. Pannu NS
    5. Steiner RA
    6. Nicholls RA
    7. Winn MD
    8. Long F
    9. Vagin AA

    (2011) REFMAC5 for the refinement of macromolecular crystal structures

    Acta Crystallographica. Section D, Biological Crystallography 67:355–367.

    https://doi.org/10.1107/S0907444911001314

    • PubMed
    • Google Scholar
30. 1. Narendra D
    2. Walker JE
    3. Youle R

    (2012) Mitochondrial quality control mediated by PINK1 and parkin: links to parkinsonism

    Cold Spring Harbor Perspectives in Biology 4:a011338.

    https://doi.org/10.1101/cshperspect.a011338

    • PubMed
    • Google Scholar
31. 1. Ordureau A
    2. Sarraf SA
    3. Duda DM
    4. Heo JM
    5. Jedrychowski MP
    6. Sviderskiy VO
    7. Olszewski JL
    8. Koerber JT
    9. Xie T
    10. Beausoleil SA
    11. Wells JA
    12. Gygi SP
    13. Schulman BA
    14. Harper JW

    (2014) Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis

    Molecular Cell 56:360–375.

    https://doi.org/10.1016/j.molcel.2014.09.007

    • PubMed
    • Google Scholar
32. 1. Riley BE
    2. Lougheed JC
    3. Callaway K
    4. Velasquez M
    5. Brecht E
    6. Nguyen L
    7. Shaler T
    8. Walker D
    9. Yang Y
    10. Regnstrom K
    11. Diep L
    12. Zhang Z
    13. Chiou S
    14. Bova M
    15. Artis DR
    16. Yao N
    17. Baker J
    18. Yednock T
    19. Johnston JA

    (2013) Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases

    Nature Communications 4:1982.

    https://doi.org/10.1038/ncomms2982

    • PubMed
    • Google Scholar
33. 1. Sauvé V
    2. Lilov A
    3. Seirafi M
    4. Vranas M
    5. Rasool S
    6. Kozlov G
    7. Sprules T
    8. Wang J
    9. Trempe JF
    10. Gehring K

    (2015) A Ubl/ubiquitin switch in the activation of Parkin

    The EMBO Journal 34:2492–2505.

    https://doi.org/10.15252/embj.201592237

    • PubMed
    • Google Scholar
34. 1. Sauvé V
    2. Sung G
    3. Soya N
    4. Kozlov G
    5. Blaimschein N
    6. Miotto LS
    7. Trempe JF
    8. Lukacs GL
    9. Gehring K

    (2018) Mechanism of parkin activation by phosphorylation

    Nature Structural & Molecular Biology 25:623–630.

    https://doi.org/10.1038/s41594-018-0088-7

    • PubMed
    • Google Scholar
35. 1. Schubert AF
    2. Gladkova C
    3. Pardon E
    4. Wagstaff JL
    5. Freund SMV
    6. Steyaert J
    7. Maslen SL
    8. Komander D

    (2017) Structure of PINK1 in complex with its substrate ubiquitin

    Nature 552:51–56.

    https://doi.org/10.1038/nature24645

    • PubMed
    • Google Scholar
36. 1. Scuderi S
    2. La Cognata V
    3. Drago F
    4. Cavallaro S
    5. D’Agata V

    (2014) Alternative splicing generates different parkin protein isoforms: evidences in human, rat, and mouse brain

    BioMed Research International 2014:690796.

    https://doi.org/10.1155/2014/690796

    • PubMed
    • Google Scholar
37. 1. Shiba-Fukushima K
    2. Imai Y
    3. Yoshida S
    4. Ishihama Y
    5. Kanao T
    6. Sato S
    7. Hattori N

    (2012) PINK1-mediated phosphorylation of the parkin ubiquitin-like domain primes mitochondrial translocation of parkin and regulates mitophagy

    Scientific Reports 2:1002.

    https://doi.org/10.1038/srep01002

    • PubMed
    • Google Scholar
38. 1. Shiba-Fukushima K
    2. Arano T
    3. Matsumoto G
    4. Inoshita T
    5. Yoshida S
    6. Ishihama Y
    7. Ryu KY
    8. Nukina N
    9. Hattori N
    10. Imai Y

    (2014) Phosphorylation of mitochondrial polyubiquitin by PINK1 promotes parkin mitochondrial tethering

    PLOS Genetics 10:e1004861.

    https://doi.org/10.1371/journal.pgen.1004861

    • PubMed
    • Google Scholar
39. 1. Spratt DE
    2. Walden H
    3. Shaw GS

    (2014) RBR E3 ubiquitin ligases: new structures, new insights, new questions

    The Biochemical Journal 458:421–437.

    https://doi.org/10.1042/BJ20140006

    • PubMed
    • Google Scholar
40. 1. Tanaka A
    2. Cleland MM
    3. Xu S
    4. Narendra DP
    5. Suen DF
    6. Karbowski M
    7. Youle RJ

    (2010) Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by parkin

    The Journal of Cell Biology 191:1367–1380.

    https://doi.org/10.1083/jcb.201007013

    • PubMed
    • Google Scholar
41. 1. Tang MY
    2. Vranas M
    3. Krahn AI
    4. Pundlik S
    5. Trempe JF
    6. Fon EA

    (2017) Structure-guided mutagenesis reveals a hierarchical mechanism of parkin activation

    Nature Communications 8:14697.

    https://doi.org/10.1038/ncomms14697

    • PubMed
    • Google Scholar
42. 1. Trempe JF
    2. Sauvé V
    3. Grenier K
    4. Seirafi M
    5. Tang MY
    6. Ménade M
    7. Al-Abdul-Wahid S
    8. Krett J
    9. Wong K
    10. Kozlov G
    11. Nagar B
    12. Fon EA
    13. Gehring K

    (2013) Structure of parkin reveals mechanisms for ubiquitin ligase activation

    Science 340:1451–1455.

    https://doi.org/10.1126/science.1237908

    • PubMed
    • Google Scholar
43. 1. Valente EM
    2. Abou-Sleiman PM
    3. Caputo V
    4. Muqit MMK
    5. Harvey K
    6. Gispert S
    7. Ali Z
    8. Del Turco D
    9. Bentivoglio AR
    10. Healy DG
    11. Albanese A
    12. Nussbaum R
    13. González-Maldonado R
    14. Deller T
    15. Salvi S
    16. Cortelli P
    17. Gilks WP
    18. Latchman DS
    19. Harvey RJ
    20. Dallapiccola B
    21. Auburger G
    22. Wood NW

    (2004) Hereditary early-onset parkinson’s disease caused by mutations in PINK1

    Science 304:1158–1160.

    https://doi.org/10.1126/science.1096284

    • PubMed
    • Google Scholar
44. 1. Walden H
    2. Rittinger K

    (2018) RBR ligase-mediated ubiquitin transfer: a tale with many twists and turns

    Nature Structural & Molecular Biology 25:440–445.

    https://doi.org/10.1038/s41594-018-0063-3

    • PubMed
    • Google Scholar
45. 1. Wang XS
    2. Cotton TR
    3. Trevelyan SJ
    4. Richardson LW
    5. Lee WT
    6. Silke J
    7. Lechtenberg BC

    (2023) The unifying catalytic mechanism of the RING-between-RING E3 ubiquitin ligase family

    Nature Communications 14:168.

    https://doi.org/10.1038/s41467-023-35871-z

    • PubMed
    • Google Scholar
46. 1. Wauer T
    2. Komander D

    (2013) Structure of the human Parkin ligase domain in an autoinhibited state

    The EMBO Journal 32:2099–2112.

    https://doi.org/10.1038/emboj.2013.125

    • PubMed
    • Google Scholar
47. 1. Wauer T
    2. Simicek M
    3. Schubert A
    4. Komander D

    (2015) Mechanism of phospho-ubiquitin-induced PARKIN activation

    Nature 524:370–374.

    https://doi.org/10.1038/nature14879

    • PubMed
    • Google Scholar
48. 1. Wenzel DM
    2. Lissounov A
    3. Brzovic PS
    4. Klevit RE

    (2011) UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids

    Nature 474:105–108.

    https://doi.org/10.1038/nature09966

    • PubMed
    • Google Scholar
49. 1. Zhuang N
    2. Li L
    3. Chen S
    4. Wang T

    (2016) PINK1-dependent phosphorylation of PINK1 and parkin is essential for mitochondrial quality control

    Cell Death & Disease 7:e2501.

    https://doi.org/10.1038/cddis.2016.396

    • PubMed
    • Google Scholar

Author details

1. Dipti Ranjan Lenka

   Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, India

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing, Performed all the biochemical and biophysical experiments, determined crystal structures, and analyzed data

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0009-8890-8331

2. Shakti Virendra Dahe

   Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, India

   Contribution

   Investigation, Methodology, Purified various proteins, SEC analysis in Figure 8, and analyzed Parkin isoforms

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0009-8909-240X

3. Odetta Antico

   MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing, Performed microscopy experiments and analyzed data

   Competing interests

   No competing interests declared

4. Pritiranjan Sahoo

   Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, India

   Contribution

   Methodology, Performed ITC measurements

   Competing interests

   No competing interests declared

5. Alan R Prescott

   Division of Cell Signalling and Immunology, Dundee Imaging Facility, School of Life Sciences, University of Dundee, Dundee, United Kingdom

   Contribution

   Methodology, Microscopy data acquisition

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0747-7317

6. Miratul MK Muqit

   MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom

   Contribution

   Formal analysis, Supervision, Funding acquisition, Project administration, Writing – review and editing, Supervised microscopy study

   Competing interests

   MM. is a member of the Scientific Advisory Board of Montara Therapeutics Inc and a scientific consultant to MSD UK

   "This ORCID iD identifies the author of this article:" 0000-0001-9733-2404

7. Atul Kumar

   Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, India

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing, Refined crystal structures and analyzed data

   For correspondence

   atul@iiserb.ac.in

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4675-6623

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