Structure of PINK1 in complex with its substrate ubiquitin

Schubert, Alexander F; Gladkova, Christina; Pardon, Els; Wagstaff, Jane L.; Freund, Stefan; Steyaert, Jan; Maslen, Sarah; Komander, David

doi:10.1038/nature24645

Cited by 141 publications

(194 citation statements)

References 64 publications

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“…For a more comprehensive mapping of the HDX‐MS results, we first analyzed the 3D structure of D337N‐TcPINK1 121–570 using small‐angle X‐ray scattering (SAXS). Two groups recently reported crystal structures of PINK1 from insects: (i) apo TcPINK1 150–570 with a partial deletion of insert 3 and a S205E mutation ; (ii) Pediculus humanus corporis (Ph)PINK1 143–575 in complex with Ub TVLN and a nanobody, with phosphorylation at site equivalent to Ser205 in TcPINK1 . The two structures were used as templates to model the structure of TcPINK1 121–570 with restraints from SAXS data for the monomer (Fig A and Appendix Fig S8).…”

Section: Resultsmentioning

confidence: 99%

PINK 1 autophosphorylation is required for ubiquitin recognition

et al. 2018

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Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in at Ser205. Small-angle X-ray scattering and hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin.

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Section: Resultsmentioning

confidence: 99%

PINK 1 autophosphorylation is required for ubiquitin recognition

et al. 2018

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“…When PINK1 transport through TOM is hindered, for instance by a depolarized mitochondria membrane potential (MMP), it accumulates on the OMM and auto‐phosphorylates at Ser228 and Ser402 . The activated PINK1 phosphorylates Ub chains at Ser65, which recruit Parkin to the OMM and activate Parkin's E3 Ub ligase activity via phosphorylation . Just as PINK1 phosphorylates Ub, PINK1 also phosphorylates Parkin directly at Ser65, signifying a secondary mode of Parkin activation .…”

Section: Parkinson's Disease Relevant Protein Kinasesmentioning

confidence: 99%

“…171 When PINK1 transport through TOM is hindered, for instance by a depolarized mitochondria membrane potential (MMP), it and Ser402. 173,174 The activated PINK1 phosphorylates Ub chains at Ser65, 175,176 which recruit Parkin to the OMM 177 and activate Parkin's E3 Ub ligase activity via phosphorylation. 178 Just as PINK1…”

Section: Pten-induced Kinase 1 (Pink1)mentioning

confidence: 99%

Phosphoregulation on mitochondria: Integration of cell and organelle responses

2019

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Mitochondria are highly integrated organelles that are crucial to cell adaptation and mitigating adverse physiology. Recent studies demonstrate that fundamental signal transduction pathways incorporate mitochondrial substrates into their biological programs. Reversible phosphorylation is emerging as a useful mechanism to modulate mitochondrial function in accordance with cellular changes. Critical serine/threonine protein kinases, such as the c‐Jun N‐terminal kinase (JNK), protein kinase A (PKA), PTEN‐induced kinase‐1 (PINK1), and AMP‐dependent protein kinase (AMPK), readily translocate to the outer mitochondrial membrane (OMM), the interface of mitochondria‐cell communication. OMM protein kinases phosphorylate diverse mitochondrial substrates that have discrete effects on organelle dynamics, protein import, respiratory complex activity, antioxidant capacity, and apoptosis. OMM phosphorylation events can be tempered through the actions of local protein phosphatases, such as mitogen‐activated protein kinase phosphatase‐1 (MKP‐1) and protein phosphatase 2A (PP2A), to regulate the extent and duration of signaling. The central mediators of OMM signal transduction are the scaffold proteins because the relative abundance of these accessory proteins determines the magnitude and duration of a signaling event on the mitochondrial surface, which dictates the biological outcome of a local signal transduction pathway. The concentrations of scaffold proteins, such as A‐kinase anchoring proteins (AKAPs) and Sab (or SH3 binding protein 5—SH3BP5), have been shown to influence neuronal survival and vulnerability, respectively, in models of Parkinson's disease (PD), highlighting the importance of OMM signaling to health and disease. Despite recent progress, much remains to be discovered concerning the mechanisms of OMM signaling. Nonetheless, enhancing beneficial OMM signaling events and inhibiting detrimental protein‐protein interactions on the mitochondrial surface may represent highly selective approaches to restore mitochondrial health and homeostasis and mitigate organelle dysfunction in conditions such as PD.

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“…The transient nature of kinase–substrate complexes not only poses a challenge to their de novo identification but also to their high‐resolution structural characterization. Relatively few structures of kinase–substrate phosphorylation complexes exist in the literature, and even fewer depict protein rather than peptide substrates . A recent structure of the kinase PINK1 in complex with its substrate P‐Ubiquitin required mutation of the kinase to stabilize binding to Ubiquitin as well as a llama antibody raised against the chemically crosslinked PINK1/Ub complex to aid in stabilization for X‐ray structure determination.…”

Section: Resultsmentioning

confidence: 99%

“…Relatively few structures of kinase-substrate phosphorylation complexes exist in the literature, and even fewer depict protein rather than peptide substrates. [27][28][29][30][31] A recent structure of the kinase PINK1 in complex with its substrate P-Ubiquitin required mutation of the kinase to stabilize binding to Ubiquitin as well as a llama antibody raised against the chemically crosslinked PINK1/Ub complex to aid in stabilization for X-ray structure determination. The SBPP probes described here produce a specific intramolecular crosslink at the phosphorylation site which could aid in isolation of 1:1 kinase:substrate complexes, improving homogeneity in samples used for cryo-EM or crystallography.…”

Section: Resultsmentioning

confidence: 99%

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

et al. 2019

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The proteomic mapping of enzyme–substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide‐based substrate analog capable of reprogramming the wild‐type phosphoryl‐transfer reaction to produce a kinase‐acrylamide‐based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP‐based methacrylate crosslinker (ATP‐MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP‐MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP‐based methacrylate (ADP‐MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP‐MA over ATP‐MA is due to reduced inhibition of the second step of the kinase–substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP‐MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site‐specific manner. The kinase–substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.

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Structure of PINK1 in complex with its substrate ubiquitin

Cited by 141 publications

References 64 publications

PINK 1 autophosphorylation is required for ubiquitin recognition

PINK 1 autophosphorylation is required for ubiquitin recognition

Phosphoregulation on mitochondria: Integration of cell and organelle responses

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

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