Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mechanism of parkin activation by phosphorylation

Nature Structural & Molecular Biology volume 25, pages 623–630 (2018)Cite this article

Subjects

A Publisher Correction to this article was published on 19 July 2018

This article has been updated

Abstract

Mutations in the ubiquitin ligase parkin are responsible for a familial form of Parkinson’s disease. Parkin and the PINK1 kinase regulate a quality-control system for mitochondria. PINK1 phosphorylates ubiquitin on the outer membrane of damaged mitochondria, thus leading to recruitment and activation of parkin via phosphorylation of its ubiquitin-like (Ubl) domain. Here, we describe the mechanism of parkin activation by phosphorylation. The crystal structure of phosphorylated Bactrocera dorsalis (oriental fruit fly) parkin in complex with phosphorylated ubiquitin and an E2 ubiquitin-conjugating enzyme reveals that the key activating step is movement of the Ubl domain and release of the catalytic RING2 domain. Hydrogen/deuterium exchange and NMR experiments with the various intermediates in the activation pathway confirm and extend the interpretation of the crystal structure to mammalian parkin. Our results rationalize previously unexplained Parkinson’s disease mutations and the presence of internal linkers that allow large domain movements in parkin.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Structure of ternary complex of activated parkin.
Fig. 2: Highlights of the activated parkin structure.
Fig. 3: Loss of the pUbl-binding site prevents parkin activation and pUbl binding.
Fig. 4: HDX–MS detection of conformational changes upon activation of rat parkin by phosphorylation and phospho-ubiquitin binding.
Fig. 5: Pathway of parkin activation.

Similar content being viewed by others

Change history

  • 19 July 2018

    In the version of this article initially published, RING2 in the schematic to the left in Fig. 1b was mislabeled as RING0. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Martin, I., Dawson, V. L. & Dawson, T. M. Recent advances in the genetics of Parkinson’s disease. Annu. Rev. Genom. Hum. Genet. 12, 301–325 (2011).

    Article  CAS  Google Scholar 

  2. Kitada, T. et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608 (1998).

    Article  PubMed  CAS  Google Scholar 

  3. Valente, E. M. et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304, 1158–1160 (2004).

    Article  PubMed  CAS  Google Scholar 

  4. Pickles, S., Vigié, P. & Youle, R. J. Mitophagy and quality control mechanisms in mitochondrial maintenance. Curr. Biol. 28, R170–R185 (2018).

    Article  PubMed  CAS  Google Scholar 

  5. Trempe, J. F. et al. Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science 340, 1451–1455 (2013).

    Article  PubMed  CAS  Google Scholar 

  6. Wauer, T. & Komander, D. Structure of the human Parkin ligase domain in an autoinhibited state. EMBO J. 32, 2099–2112 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Riley, B. E. et al. Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases. Nat. Commun. 4, 1982 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Geisler, S. et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12, 119–131 (2010).

    Article  PubMed  CAS  Google Scholar 

  9. Matsuda, N. et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J. Cell Biol. 189, 211–221 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 8, e1000298 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Vives-Bauza, C. et al. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc. Natl Acad. Sci. USA 107, 378–383 (2010).

    Article  PubMed  Google Scholar 

  12. Kane, L. A. et al. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J. Cell Biol. 205, 143–153 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Kazlauskaite, A. et al. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem. J. 460, 127–139 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Koyano, F. et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510, 162–166 (2014).

    Article  PubMed  CAS  Google Scholar 

  15. Ordureau, A. et al. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol. Cell 56, 360–375 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Sauvé, V. et al. A Ubl/ubiquitin switch in the activation of Parkin. EMBO J. 34, 2492–2505 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Kazlauskaite, A. et al. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation. EMBO Rep. 16, 939–954 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Wauer, T., Simicek, M., Schubert, A. & Komander, D. Mechanism of phospho-ubiquitin-induced PARKIN activation. Nature 524, 370–374 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Kumar, A. et al. Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis. EMBO J. 34, 2506–2521 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Sugiura, A., McLelland, G. L., Fon, E. A. & McBride, H. M. A new pathway for mitochondrial quality control: mitochondrial-derived vesicles. EMBO J. 33, 2142–2156 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Matheoud, D. et al. Parkinson’s disease-related proteins PINK1 and Parkin repress mitochondrial antigen presentation. Cell 166, 314–327 (2016).

    Article  PubMed  CAS  Google Scholar 

  22. Wenzel, D. M., Lissounov, A., Brzovic, P. S. & Klevit, R. E. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105–108 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Seirafi, M., Kozlov, G. & Gehring, K. Parkin structure and function. FEBS J. 282, 2076–2088 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. McGinty, R. K., Henrici, R. C. & Tan, S. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514, 591–596 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Gladkova, C., Maslen, S., Skehel, J.M. & Komander, D. Mechanism of parkin activation by PINK1. Nature https://doi.org/10.1038/s41586-018-0224-x (2018).

  27. Swatek, K. N. & Komander, D. Ubiquitin modifications. Cell Res. 26, 399–422 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Spratt, D. E. et al. A molecular explanation for the recessive nature of parkin-linked Parkinson’s disease. Nat. Commun. 4, 1983 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Hattori, N. et al. Point mutations (Thr240Arg and Gln311Stop) [correction of Thr240Arg and Ala311Stop] in the Parkin gene. Biochem. Biophys. Res. Commun. 249, 754–758 (1998).

    Article  PubMed  CAS  Google Scholar 

  30. Shimura, H. et al. Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nat. Genet. 25, 302–305 (2000).

    Article  PubMed  CAS  Google Scholar 

  31. Periquet, M. et al. Parkin mutations are frequent in patients with isolated early-onset parkinsonism. Brain 126, 1271–1278 (2003).

    Article  PubMed  Google Scholar 

  32. Tang, M. Y. et al. Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation. Nat. Commun. 8, 14697 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Chaugule, V. K. et al. Autoregulation of Parkin activity through its ubiquitin-like domain. EMBO J. 30, 2853–2867 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. McLelland, G. L., Soubannier, V., Chen, C. X., McBride, H. M. & Fon, E. A. Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control. EMBO J. 33, 282–295 (2014).

    PubMed  PubMed Central  CAS  Google Scholar 

  35. Abbas, N. et al. A wide variety of mutations in the parkin gene are responsible for autosomal recessive parkinsonism in Europe. Hum. Mol. Genet. 8, 567–574 (1999).

    Article  PubMed  CAS  Google Scholar 

  36. Pankratz, N. et al. Parkin dosage mutations have greater pathogenicity in familial PD than simple sequence mutations. Neurology 73, 279–286 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Safadi, S. S. & Shaw, G. S. A disease state mutation unfolds the parkin ubiquitin-like domain. Biochemistry 46, 14162–14169 (2007).

    Article  PubMed  CAS  Google Scholar 

  38. Aguirre, J. D., Dunkerley, K. M., Mercier, P. & Shaw, G. S. Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation. Proc. Natl Acad. Sci. USA 114, 298–303 (2017).

    Article  PubMed  CAS  Google Scholar 

  39. Steffen, J. D., McCauley, M. M. & Pascal, J. M. Fluorescent sensors of PARP-1 structural dynamics and allosteric regulation in response to DNA damage. Nucleic Acids Res 44, 9771–9783 (2016).

    PubMed  PubMed Central  CAS  Google Scholar 

  40. Ordureau, A. et al. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc. Natl Acad. Sci. USA 112, 6637–6642 (2015).

    Article  PubMed  CAS  Google Scholar 

  41. Okiyoneda, T. et al. Mechanism-based corrector combination restores ΔF508-CFTR folding and function. Nat. Chem. Biol. 9, 444–454 (2013).

    Article  PubMed  CAS  Google Scholar 

  42. Calles-Garcia, D. et al. Single-particle electron microscopy structure of UDP-glucose:glycoprotein glucosyltransferase suggests a selectivity mechanism for misfolded proteins. J. Biol. Chem. 292, 11499–11507 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Duda, D. M. et al. Structure of HHARI, a RING-IBR-RING ubiquitin ligase: autoinhibition of an Ariadne-family E3 and insights into ligation mechanism. Structure 21, 1030–1041 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Kelsall, I. R. et al. TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes. EMBO J. 32, 2848–2860 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Ho, S. R., Lee, Y. J. & Lin, W. C. Regulation of RNF144A E3 ubiquitin ligase activity by self-association through its transmembrane domain. J. Biol. Chem. 290, 23026–23038 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Smit, J. J. et al. The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension. EMBO J. 31, 3833–3844 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Stieglitz, B., Morris-Davies, A. C., Koliopoulos, M. G., Christodoulou, E. & Rittinger, K. LUBAC synthesizes linear ubiquitin chains via a thioester intermediate. EMBO Rep. 13, 840–846 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Pao, K. C. et al. Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation. Nat. Chem. Biol. 12, 324–331 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Lazarou, M. et al. PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding. J. Cell Biol. 200, 163–172 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Dove, K. K. et al. Structural studies of HHARI/UbcH7~Ub reveal unique E2~Ub conformational restriction by RBR RING1. Structure 25, 890–900.e5 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Rasool, S. et al. PINK1 autophosphorylation is required for ubiquitin recognition. EMBO Rep. 19, e44981 (2018).

    Article  PubMed  CAS  Google Scholar 

  52. Caulfield, T. R. et al. Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin. PLOS Comput. Biol. 10, e1003935 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Ordureau, A. et al. Dynamics of PARKIN-dependent mitochondrial ubiquitylation in induced neurons and model systems revealed by digital snapshot proteomics. Mol. Cell 70, 211–227.e8 (2018).

    Article  PubMed  CAS  Google Scholar 

  54. Geisler, S., Vollmer, S., Golombek, S. & Kahle, P. J. The ubiquitin-conjugating enzymes UBE2N, UBE2L3 and UBE2D2/3 are essential for Parkin-dependent mitophagy. J. Cell Sci. 127, 3280–3293 (2014).

    Article  PubMed  CAS  Google Scholar 

  55. Dove, K. K. & Klevit, R. E. RING-between-RING E3 ligases: emerging themes amid the variations. J. Mol. Biol. 429, 3363–3375 (2017).

    Article  PubMed  CAS  Google Scholar 

  56. Lechtenberg, B. C. et al. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation. Nature 529, 546–550 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Yuan, L., Lv, Z., Atkison, J. H. & Olsen, S. K. Structural insights into the mechanism and E2 specificity of the RBR E3 ubiquitin ligase HHARI. Nat. Commun. 8, 211 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Martino, L., Brown, N. R., Masino, L., Esposito, D. & Rittinger, K. Determinants of E2-ubiquitin conjugate recognition by RBR E3 ligases. Sci. Rep. 8, 68 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Cunningham, C. N. et al. USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria. Nat. Cell Biol. 17, 160–169 (2015).

    Article  PubMed  CAS  Google Scholar 

  60. Sarraf, S. A. et al. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496, 372–376 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Kabsch, W. Xds. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  62. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D. Biol. Crystallogr. 68, 352–367 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D. Biol. Crystallogr. 65, 582–601 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  PubMed  CAS  Google Scholar 

  68. Bai, Y., Milne, J. S., Mayne, L. & Englander, S. W. Primary structure effects on peptide group hydrogen exchange. Proteins 17, 75–86 (1993).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. M. Pascal (Université de Montréal) for the Staphylococcus aureus sortase A construct and M. Seirafi for preparation of proteins for NMR spectroscopy. We thank the Canadian Light Source for access and data collection on the CMCF beamline 08ID-1, and group members for insightful and helpful discussions. We acknowledge support from the Michael J. Fox Foundation and Canadian Institutes of Health Research.

Author information

Author notes

  1. These authors contributed equally: Véronique Sauvé, George Sung.

Authors and Affiliations

  1. Department of Biochemistry, McGill University, Montreal, Quebec, Canada

    Véronique Sauvé, George Sung, Guennadi Kozlov, Nina Blaimschein, Lis Schwartz Miotto & Kalle Gehring

  2. McGill Centre for Structural Biology, McGill University, Montreal, Quebec, Canada

    Véronique Sauvé, George Sung, Naoto Soya, Guennadi Kozlov, Nina Blaimschein, Lis Schwartz Miotto, Jean-François Trempe, Gergely L. Lukacs & Kalle Gehring

  3. Department of Physiology, McGill University, Montreal, Quebec, Canada

    Naoto Soya & Gergely L. Lukacs

  4. Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

    Jean-François Trempe

Authors
  1. Véronique Sauvé
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. George Sung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Naoto Soya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guennadi Kozlov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nina Blaimschein
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lis Schwartz Miotto
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jean-François Trempe
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gergely L. Lukacs
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kalle Gehring
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.S. designed constructs, froze crystals, performed autoubiquitination assays and prepared HDX samples. V.S. and G.S. performed crystallization and crystal-structure refinement. G.S. performed sortase A reactions. N.S. performed HDX experiments and analysis. G.K., L.M., and N.B. performed NMR experiments. J.-F.T. collected X-ray data, performed phasing and crystal-structure refinement, and analyzed E2-parkin interactions. V.S., G.S., J.-F.T. and K.G. wrote the manuscript. G.L. and K.G. oversaw the project.

Corresponding authors

Correspondence to Jean-François Trempe or Kalle Gehring.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Sequence alignments of parkin orthologs and E2 enzymes.

a, Sequence alignment of parkin orthologs from Bactrocera dorsalis (BD), Rattus norvegicus (RN) and Homo sapiens (HS). b, Sequence alignment of human E2 ubiquitin-conjugating enzymes. The first five E2 enzymes have been reported to function with parkin in vitro or in cells. Blue shading indicates sequence conservation. Residues in the parkin-UbcH7 interface are labeled in green. Residues in the pUbl-RING0 interface are labeled in red. The catalytic cysteines are labeled in yellow

Supplementary Figure 2 Autoubiquitination assay and crystal structures of phosphorylated Bactrocera parkin fused to UbcH7, in the presence of pUb.

a, The autoubiquitination activity of UbcH7-parkin fusion proteins was tested with phosphorylated proteins in the presence of Ub and visualized on SDS-PAGE gel (n = 1). The first two lanes show the activity of UbcH7 and parkin alone as a control. The third to sixth lanes show the activity of the UbcH7-parkin fusions with different length linkers. The linker length between UbcH7 and parkin is 8, 10, 13, 15 amino acids. b, The structure with the REP linker and RING2 present was solved to 4.8 Å using molecular replacement and zinc SAD. Anomalous map was generated from data collected for UbcH7-phosphorylated parkin/pUb crystal at 1.28 Å wavelength. The anomalous dispersion (light grey mesh) confirms the position of zinc atoms (black spheres) of RING0 (green), RING1 (cyan) and IBR (magenta) in the crystal structure of UbcH7-phosphorylated parkin+pUb. Zinc atoms of symmetry-related UbcH7-phosphorylated parkin+pUb molecules within the crystal are displayed in light orange. No unassigned anomalous density was observed. c, The structure without the disordered elements was solved to 3.8 Å using molecular replacement using 4.8 Å structure as the model. The all-atom root mean square deviation between the two structures is 0.2 Å

Supplementary Figure 3 Steric interactions regulate parkin activity.

a, Overlay of activated Bactrocera parkin structure (colored) with the RING2 modeled from the inactive parkin (grey, PDB 4K95). The C-terminal helix of RING2 occupies the same position as pUbl residues preceding the phosphoserine residue (pSer94). b, Overlay of UbcH7 with the Ubl domain from inactive parkin. The Ubl domain prevents UbcH7 residues Glu60 and Phe63 from binding to residues Gln301, Tyr302, Ser305, and Arg 306 of helix 1 of the parkin RING1 domain. c, Overlay of UbcH7 with the REP linker from inactive parkin. The REP residues preceding the helix block contact of UbcH7 residues Lys96, Pro97, and Ala98 with Bactrocera parkin residues Ala326 and Leu274

Supplementary Figure 4 Electrostatic interactions between UbcH7 and parkin RING1.

a, Structure of Bactrocera parkin bound to UbcH7. An electrostatic potential map was calculated for the isolated RING1 domain. The side-chains of Arg5 and Arg6 in UbcH7 interact with a negatively charged surface in RING1. b, Model of rat Parkin (PDB 4ZYN) bound to UbcH7 with an electrostatic potential map computed for the isolated RING1 domain (REP and Ubl removed)

Supplementary Figure 5 Phosphorylation of parkin mutants for ubiquitin assays.

a, For the autoubiquitination assay, the mutants and WT were phosphorylated and verified on a 7.5% phos-tag SDS-PAGE (n = 1 assay). b, For the complementation assay, the mutants and WT were phosphorylated and verified on a 7.5 % phos-tag SDS-PAGE (n = 1 assay). c, Intact mass spectrometry of the phosphorylated proteins, collected on a Impact II ESI-QTOF (n = 1 assay). The predicted average mass is displayed on the left.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5

Reporting Summary

Supplementary Dataset 1

Uncropped NMR spectra and gel from Figure 3

Supplementary Dataset 2

Graphic presentation of HDX-MS data from Figure 4

Supplementary Dataset 3

Excel data of HDX-MS data from Figure 4

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sauvé, V., Sung, G., Soya, N. et al. Mechanism of parkin activation by phosphorylation. Nat Struct Mol Biol 25, 623–630 (2018). https://doi.org/10.1038/s41594-018-0088-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing