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

Riley, Brigit E.; Lougheed, Julie C.; Callaway, Kari; Velasquez, M.; Brecht, E.; Nguyen, Lan K.; Shaler, Thomas A.; Walker, Duncan; Yang, Yanli; Regnström, Karin; Diep, Linnea; Zhang, Z.; Chiou, Shyh‐Horng; Bova, Michael P.; Artis, Dean R.; Yao, Nanhua; Baker, Jeanne; Yednock, Ted; Johnston, Jennifer

doi:10.1038/ncomms2982

Cited by 290 publications

(386 citation statements)

References 52 publications

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“…These differences when pUBL is bound, especially in residues G385-E395, suggest these are specifically reporting the phosphate moiety and indicate that pSer65 is likely near this portion of the tether. Notably, we do not observe chemical shift changes beyond R396, a region that includes W403 and is proposed to suppress ligase activity by blocking the E2 binding site (35,36). Therefore, it is conceivable that UBL phosphorylation serves a dual purpose: It optimizes the pUb-binding site (15) and primes the upstream region of the tether for remodeling to accommodate the incoming E2-Ub conjugate.…”

Section: Significancementioning

confidence: 91%

See 1 more Smart Citation

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

Aguirre

Dunkerley

Mercier

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson's disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTENinduced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin's UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2-ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin's ubiquitin ligase potential. . Multiple studies show that in response to mitochondrial oxidative stress parkin activity is stimulated through phosphorylation by PINK1 (4, 5). This in turn facilitates parkin-mediated ubiquitination of several proteins at the outer mitochondrial membrane and signals the turnover of damaged mitochondria through the mitophagy pathway (6-8). Some mutations in parkin cause its dysfunction, leading to an accumulation of mitochondrial damage that appears to be especially detrimental in neurons, a potential cause for Parkinson's disease.Parkin belongs to the RBR subfamily of E3 ubiquitin ligases (9) that function by transferring Ub from an E2-conjugating enzyme to a substrate through an E3-Ub intermediate (10). These enzymes are structurally autoinhibited in their native states by unique accessory domains (11-13), indicating that RBR ligases must be activated to carry out their full ubiquitination potential. Specifically, parkin contains an N-terminal ubiquitin-like (UBL) domain shown to inhibit Ub ligase activity (11). Threedimensional structures of parkin show UBL associates with the C-terminal region (R0RBR) through both ionic and hydrophobic interactions, blocking the proposed E2 recognition site (14, 15). PINK1 stimulates parkin activity through phosphorylation of both Ub and parkin's UBL domain at an equivalent serine 65 position in sequence and structure (16)(17)(18)(19)(20)(21). Whereas each phosphorylation event can increase parkin activity independently, maximal activity is obtained when both parkin and Ub are phosphorylated (18,19). Phosphorylation of parkin at S65 increases parkin's affinity for phosphoubiquitin (pUb) (1...

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Section: Significancementioning

confidence: 91%

“…3D). Much of this region is flexible based on heteronuclear NOE experiments (15) and is unresolved in crystal structures (15,35). Chemical shift perturbations show that a portion of this tether (residues F381-E385) lies near the UBL domain in the autoinhibited state.…”

Section: Significancementioning

confidence: 99%

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

Aguirre

Dunkerley

Mercier

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…What is less clear is how parkin positions the E2~Ub conjugate to enable transfer of the Ub molecule to the RING2(Rcat) domain as a necessary step for catalysis. Current crystal structures of parkin show the proposed E2 binding site on the RING1 domain is > 50 Å from the catalytic site (C431) in the RING2(Rcat) domain suggesting a significant conformational rearrangement is needed (Riley et al , 2013; Trempe et al , 2013; Wauer & Komander, 2013; Kumar et al , 2015, 2017; Sauvé et al , 2015; Wauer et al , 2015). A similar dilemma arises from recent structures of HHARI in complex with UbcH7‐Ub that show the ubiquitin molecule is 47–53 Å from the catalytic site (Dove et al , 2017; Yuan et al , 2017).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early‐onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N‐terminal ubiquitin‐like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho‐ubiquitin‐loaded C‐terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re‐modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.

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“…Once on the OMM, PINK1 kinase activity recruits Parkin from the cytosol (8,9). Although Parkin adopts a self-inhibited conformation in solution (23)(24)(25), it becomes fully activated in a PINK1-dependent manner on the mitochondria (9,21). Parkin ubiquitinates numerous proteins of the OMM (26,27), and thereby recruits autophagy-related proteins to the damaged mitochondrion for autophagosome assembly (28)(29)(30).…”

mentioning

confidence: 99%

Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility

Shlevkov

Kramer

Schapansky

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

126

119

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The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy.is the second most common neurodegenerative disorder, and is closely linked to mitochondrial dysfunction (1, 2). Two hereditary forms of recessive PD are caused by mutations in PINK1 (PTEN-induced putative kinase 1), a Ser/Thr mitochondrial kinase, and Parkin, a cytosolic E3 ubiquitin ligase (3, 4). The realization that these proteins are in a single pathway, with PINK1 acting upstream of Parkin to influence mitochondrial properties, was a critical step in uncovering the underlying pathological mechanisms of PD (5-7). This pathway can trigger the selective autophagy of damaged mitochondria, termed mitophagy (8, 9), but additional cellular functions have also been indicated for PINK1 and Parkin (10-15). Much, however, remains unclear about how the PINK1/Parkin pathway is regulated.In current models of PINK1/Parkin mitophagy (reviewed in ref. 1), healthy mitochondria import a PINK1 precursor constitutively to the inner membrane, where it is cleaved (16-18). The cleaved form then returns to the cytoplasm and is degraded by the N-end rule pathway (19). Mitochondrial depolarization, or protein misfolding in the matrix of energized mitochondria (20), prevent the import and degradation of PINK1, resulting in the accumulation of PINK1 on the outer mitochondrial membrane (OMM) (9,21,22). Once on the OMM, PINK1 kinase activity recruits Parkin from the cytosol (8, 9). Although Parkin adopts a self-inhibited conformation in solution (23-25), it becomes fully activated in a PINK1-dependent manner on the mitochondria (9, 21). Parkin ubiquitinates numerous proteins of the OMM (26, 27), and thereby recruits autophagy-related proteins to the damaged mitochondrion for autophagosome assembly (28-30).How PINK1 recruits and activa...

show abstract

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

Cited by 290 publications

References 52 publications

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

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

Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility

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