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Forschungsarbeit

The structure of human Parkin –
What a member of a new E3 ligase class reveals
about Parkinson’s disease

Von Tobias Wauer (26.05.2014)

No matter if the cell divides, triggers an immune response or degrades cellular waste, all of these processes and many more are regulated by a small protein tag called ubiquitin. Therefore it is no surprise that a malfunction in ubiquitination has serious effects on human health as examples in cancer and neurodegenerative disease show. In functional cells ubiquitin is transferred onto the substrate usually forming poly-ubiquitin chains, a process that is mediated by E3 ligase proteins. The over 600 E3 ligases can be classified into RING E3s, HECT E3s and newly described RING-HECT hybrid ligases called RING-between-RING (RBR) E3 ligase class. [1] For my PhD at he MRC Laboratory of Molecular Biology in Cambridge I set out to understand this poorly described RBR E3 ligase family and determined the molecular structure of the RBR E3 ligase Parkin, a key factor in the understanding of Parkinson’s disease.

Wauer: Fig. 1[Bildunterschrift / Subline]: Figure 1: Parkin ubiquitination of mitochondria during mitophagy. Parkin comprises the Ubl-domain (green), the Unique Parkin Domain (UPD in blue), and the RBR motif consisting of RING1 (cyan), IBR (purple) and RING2-domain (orange). The loaded E2 is bound to the RING1 domain and ubiquitin is discharged to form a covalent intermediate with the active site cysteine of Parkin in the RING2 domain. From there ubiquitin is transferred onto damaged mitochondria and causes the organelle to be degraded.

Parkinson’s disease is an incurable neurodegenerative disorder characterized by the loss of dopaminergic neurons. Genetic predisposition has been identified as an important factor for the disease and mutations in the E3 ligase Parkin are the most frequent cause of early onset Parkinson’s disease. [2] Parkin is a crucial component of the cellular homeostasis and mediates a process called mitophagy in which mitochondria, the “power plants” of the cells, get degraded upon damage. In this poorly understood process, Parkin is recruited to faulty mitochondria, accepts ubiquitin onto its active site cysteine and transfers it onto substrates in the mitochondrial membrane, which marks them for degradation (Fig. 1). If this process is impaired, toxic components leak from the damaged mitochondria and neuronal cell death eventually leads to Parkinson’s disease. [3,4]

In the first half of my PhD I used a combination of molecular biology, biochemistry, and x-ray crystallography to determine the structure of human Parkin, which was together with several other structures published at the same time, among the first RBR E3 ligase structures solved to date (Fig 2) [5-8]. This not only revealed completely unknown folds for some of the domains comprising the protein, but to our great surprise also showed that Parkin is trapped in an auto-inhibited conformation. In a fully active protein one would expect that an E2 conjugating enzyme upon binding to Parkin, transfers the ubiquitin onto the active cysteine situated in the RING2 domain (Fig. 3). However in Parkin this transfer is rendered inhibited by 2 mechanisms: On the one hand access to the active site of Parkin is impaired by the domain we termed Unique Parkin Domain (UPD) (blue in Fig. 2) and on the other hand a linker helix (red in fig. 2) blocks the docking site for the E2 delivering the ubiquitin. Indeed, a construct lacking the inhibitory UPD exhibited increased Parkin activity. Another interesting surprise was to find the ubiquitin acceptor residue to be embedded in an unprecedented “catalytic triad”, an amino acid constellation that renders the catalytic cysteine more reactive. Hence mutating these residues decreased enzyme activity.

[Bildunterschrift / Subline]: Figure 2: Structure of Parkin with Parkinson’s Disease patient mutations on inhibitory interfaces. The construct of human Parkin used for crystallization comprises the UPD (blue), the RING1-domain (cyan), the IBR-domain (purple), the linker helix (red) and the RING2-domain (orange) with the active site. Hot-spots of Parkinson’s Disease patient mutations can be found at the auto-inhibitory interface between the UPD and RING2-domain (left box), and between the linker helix and RING1 domain (right box). Mutated amino acids are shown in ball-and-stick representation.

Intriguingly we could map 57 Parkinson’s disease patient mutations on my structure and for the first time were able to categorize many of them according to their mechanism of action. Some point mutations disrupt the protein fold and destabilize the protein whereas others interfered with the catalytic mechanism for example by disrupting active site residues. To our amazement two hot spots of patient mutations were situated at the interfaces trapping Parkin in an autoinhibited conformation, namely between the UPD and the RING2 domain, and between the linker helix and the RING1 domain respectively (Fig. 2). This indicates that some patient mutations in these regions are actually not loss-of-function mutations as previously anticipated but might be gain-of-function mutations. Experimental data indicated that disrupting these auto-inhibitory interfaces indeed increases Parkin auto-ubiquitination activity. However the involvement of increased Parkin activity in disease needs further investigation.

For the remainder of my PhD I am interested in the question how Parkin gets activated to fulfill its “housekeeping “ function in the cell, i.e. how is it transformed from a locked-up auto-inhibited state to a conformation that is able to accept and transfer ubiquitin onto damaged mitochondria (Fig. 3). In agreement with data from other labs we found the kinase PINK1, another protein that has been implicated in Parkinson’s disease, to be a potential activator of Parkin. Intriguingly it became clear that PINK1 actually phosphorylates ubiquitin itself, but how this mediates Parkin regulation needs to be further investigated.

Taken together, my work on the structure of Parkin and its mechanism of action shed new light on the underlying principles of Parkinson’s disease and may be a step towards a treatment of this incurable disease.

[Bildunterschrift / Subline]: Figure 3: Potential Parkin activation. During activation the inactive state of Parkin (left) has to undergo conformational rearrangement allowing E2 binding to the RING1-domain and ubiquitin transfer onto the active site in the RING2 domain (right).

[1] 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).
[2] Corti, O., Lesage, S. & Brice, A. What Genetics Tells us About the Causes and Mechanisms of Parkinson's Disease. Physiological Reviews 91, 1161–1218 (2011).
[3] Henchcliffe, C. & Beal, M. F. Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis. Nat Clin Pract Neurol 4, 600–609 (2008).
[4]Youle, R. J. & Narendra, D. P. Mechanisms of mitophagy. Nat Rev Mol Cell Biol 12, 9–14 (2011).
[5] Wauer, T. & Komander, D. Structure of the human Parkin ligase domain in an autoinhibited state. EMBO J (2013). doi:10.1038/emboj.2013.125
[6] Riley, B. E. et al. Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases. Nature Communications 4, 1982 (2013).
[7] Trempe, J.-F. et al. Structure of Parkin Reveals Mechanisms for Ubiquitin Ligase Activation. Science (2013). doi:10.1126/science.1237908
[8] 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 (2013). doi:10.1016/j.str.2013.04.019


mailto: Tobias Wauer
Tobias Wauer
* 1986

Scientific career
  • 2011
  • University of Cambridge (UK) Trinity College PhD in Molecular Biology MRC Laboratory of Molecular Biology with Dr. David Komander
  • 2010-2011
  • University of Oxford (UK), Master’s thesis with Prof. Hagan Bayley
  • 2005-2011
  • Technische Universität München, M. Sc./B. Sc. in Molecular Biotechnology/Biochemistry

Scholarships and Awards
  • * “LMB Cambridge Scholarships” of the MRC Laboratory of Molecular Biology
  • * German National Academic Foundation (Studienstiftung des deutschen Volkes) Scholarship
  • * Max Weber-Program of the State of Bavaria Scholarship
  • * TUM: Junge Akademie Scholarship
  • * SYMBLS conference Best Poster Prize
  • * Gold medalist iGEM competition (Team TUM)

Publications (Excerpt)
  • * Wauer, T., Gerlach, H., Mantri, S., Hill, J., Bayley, H., & Sapra, K. T. (2014). Construction and manipulation of functional three-dimensional droplet networks. ACS Nano, 8(1), 771–779. doi:10.1021/nn405433y
  • * Wauer, T., & Komander, D. (2013). Structure of the human Parkin ligase domain in an autoinhibited state. The EMBO Journal. doi:10.1038/emboj.2013.125
  • * Keusekotten, K., Elliott, P. R., Glockner, L., Fiil, B. K., Damgaard, R. B., Kulathu, Y., et al. (2013). OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell, 153(6), 1312–1326. doi:10.1016/j.cell.2013.05.014