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

1] Elan Pharmaceuticals, 180 Oyster Point Boulevard, South San Francisco, California 94080, USA [2].
Nature Communications (Impact Factor: 11.47). 06/2013; 4:1982. DOI: 10.1038/ncomms2982
Source: PubMed


Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed 'RING/HECT hybrid' enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin's cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.

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Available from: Brigit E Riley, Jul 12, 2014
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    • "However, loss of protein stability (insolubility) as we previously demonstrated [24], leads to parkin accumulation and degradation via autophagy, suggesting that parkin ubiquitination leads to protein stability. These data are congruent with parkin structure, which contains 9 potential ubiquitination sites on Lys residues [43], and its function as a hybrid RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) enzyme [43], [44], [45], [46], [47], [48], suggesting that ubiquitination may sequentially lead to parkin activation, substrate recruitment and subsequent proteasomal degradation of parkin and its substrate. Parkin is present in an auto-inhibited and inactive form usually [49] due to the complexity of its structure [43], [50], [51], however mutations may alter its allosteric conformation, leading to protein instability and affect enzymatic activity [43], [50], [51]. "
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    ABSTRACT: Parkinson's disease (PD) is a movement disorder associated with genetic and age related causes. Although autosomal recessive early onset PD linked to parkin mutations does not exhibit α-Synuclein accumulation, while autosomal dominant and sporadic PD manifest with α-Synuclein inclusions, loss of dopaminergic substantia nigra neurons is a common denominator in PD. Here we show that decreased parkin ubiquitination and loss of parkin stability impair interaction with Beclin-1 and alter α-Synuclein degradation, leading to death of dopaminergic neurons. Tyrosine kinase inhibition increases parkin ubiquitination and interaction with Beclin-1, promoting autophagic α-Synuclein clearance and nigral neuron survival. However, loss of parkin via deletion increases α-Synuclein in the blood compared to the brain, suggesting that functional parkin prevents α-Synuclein release into the blood. These studies demonstrate that parkin ubiquitination affects its protein stability and E3 ligase activity, possibly leading to α-Synuclein sequestration and subsequent clearance.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "It is noteworthy to highlight that we have previously also proposed a similar model of parkin activity repression, although we found that the unique region of parkin (i.e. between Ubl and RING1) serves as the inhibitory domain [32]. Recently, several groups have reported the crystal structure of parkin that supports our model of parkin catalytic inhibition [33–35]. Collectively, these groups found that parkin is normally kept in auto-inhibited state by two key mechanisms – (i) a linker region between IBR and RING2 that is positioned in a configuration that would block the conserved E2 ~ Ub binding site of RING1 (thus denying access to E2), and (ii) an interface that forms between the unique region of parkin and RING2 that buries the catalytic site of RING2. "
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    ABSTRACT: Disruption of the ubiquitin-proteasome system, which normally identifies and degrades unwanted intracellular proteins, is thought to underlie neurodegeneration. Supporting this, mutations of Parkin, a ubiquitin ligase, are associated with autosomal recessive parkinsonism. Remarkably, Parkin can protect neurons against a wide spectrum of stress, including those that promote proteasome dysfunction. Although the mechanism underlying the preservation of proteasome function by Parkin is hitherto unclear, we have previously proposed that Parkin-mediated K63-linked ubiquitination (which is usually uncoupled from the proteasome) may serve to mitigate proteasomal stress by diverting the substrate load away from the machinery. By means of linkage-specific antibodies, we demonstrated here that proteasome inhibition indeed promotes K63-linked ubiquitination of proteins especially in Parkin-expressing cells. Importantly, we further demonstrated that the recruitment of Ubc13 (an E2 that mediates K63-linked polyubiquitin chain formation exclusively) by Parkin is selectively enhanced under conditions of proteasomal stress, thus identifying a mechanism by which Parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress. This mode of ubiquitination appears to facilitate the subsequent clearance of Parkin substrates via autophagy. Consistent with the proposed protective role of K63-linked ubiquitination in times of proteolytic stress, we found that Ubc13-deficient cells are significantly more susceptible to cell death induced by proteasome inhibitors compared to their wild type counterparts. Taken together, our study suggests a role for Parkin-mediated K63 ubiquitination in maintaining cellular protein homeostasis, especially during periods when the proteasome is burdened or impaired.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "The structure shows the conserved phenylalanine -3 residues from the C-terminus of parkin (F463, equivalent to Y387 in HHARI) is involved in interactions with the RING0 domain. These interactions can be relieved when F463 is substituted with a tyrosine [40] leading to increased ubiquitination or covalent modification using a vinyl-sulfone probe. Likewise, the substitution of the final three residues of parkin containing F463 have been shown to be integral for proper folding and enzyme activity [39]. "
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    ABSTRACT: The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn(2+)-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.
    Full-text · Article · Aug 2013 · PLoS ONE
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