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• W4225289119 abstract "Article2 May 2022Open Access Transparent process Structural basis for feedforward control in the PINK1/Parkin pathway Véronique Sauvé Véronique Sauvé orcid.org/0000-0002-5981-4573 Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author George Sung George Sung orcid.org/0000-0002-3146-6095 Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology Search for more papers by this author Emma J MacDougall Emma J MacDougall McGill Parkinson Program, Neurodegenerative Diseases Group, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology Search for more papers by this author Guennadi Kozlov Guennadi Kozlov orcid.org/0000-0002-7742-6558 Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Anshu Saran Anshu Saran Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: ​Investigation Search for more papers by this author Rayan Fakih Rayan Fakih orcid.org/0000-0002-4328-618X Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: ​Investigation Search for more papers by this author Edward A Fon Edward A Fon orcid.org/0000-0002-5520-6239 McGill Parkinson Program, Neurodegenerative Diseases Group, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada Contribution: Supervision, Funding acquisition, Project administration Search for more papers by this author Kalle Gehring Corresponding Author Kalle Gehring [email protected] orcid.org/0000-0001-6500-1184 Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: Conceptualization, Supervision, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Véronique Sauvé Véronique Sauvé orcid.org/0000-0002-5981-4573 Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author George Sung George Sung orcid.org/0000-0002-3146-6095 Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology Search for more papers by this author Emma J MacDougall Emma J MacDougall McGill Parkinson Program, Neurodegenerative Diseases Group, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology Search for more papers by this author Guennadi Kozlov Guennadi Kozlov orcid.org/0000-0002-7742-6558 Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Anshu Saran Anshu Saran Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: ​Investigation Search for more papers by this author Rayan Fakih Rayan Fakih orcid.org/0000-0002-4328-618X Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: ​Investigation Search for more papers by this author Edward A Fon Edward A Fon orcid.org/0000-0002-5520-6239 McGill Parkinson Program, Neurodegenerative Diseases Group, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada Contribution: Supervision, Funding acquisition, Project administration Search for more papers by this author Kalle Gehring Corresponding Author Kalle Gehring [email protected] orcid.org/0000-0001-6500-1184 Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada Contribution: Conceptualization, Supervision, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Véronique Sauvé1, George Sung1,3, Emma J MacDougall2, Guennadi Kozlov1, Anshu Saran1,4, Rayan Fakih1, Edward A Fon2 and Kalle Gehring *,1 1Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada 2McGill Parkinson Program, Neurodegenerative Diseases Group, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada 3Present address: Department of Neurology and Neurosurgery and Montreal Neurological Institute, McGill University, Montreal, QC, Canada 4Present address: Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada *Corresponding author. Tel: +1-514-398-7287; E-mail: [email protected] The EMBO Journal (2022)41:e109460https://doi.org/10.15252/embj.2021109460 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson’s disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observed that phospho-ubiquitin can bind to two distinct sites on parkin, a high-affinity site on RING1 that controls parkin localization and a low-affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho-ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development. Synopsis The ubiquitin ligase parkin protects against Parkinson’s disease by binding and ubiquitinating damaged mitochondria. Here, cellular, biochemical and biophysical results identify a novel feedforward mechanism involving direct parkin activation without phosphorylation by the protein kinase PINK1. Parkin has two binding sites for phosphorylated ubiquitin (pUb) The first binding site recruits parkin to damaged mitochondria The second site binds either pUb or the phosphorylated parkin Ubl domain, to switch on ubiquitin ligase activity Introduction Parkinson’s disease is one of the most common neurodegenerative diseases. It causes motor symptoms due to the loss of dopaminergic neurons of the substantia nigra in the midbrain. Over 90% of the cases are sporadic and occur late in life. However, 5–10% of the cases are attributed to autosomal mutations that induce the disease in younger patients (Koros et al, 2017). Many of these mutations are found in PARK2 (Kitada et al, 1998) and PARK6 (Valente et al, 2004) genes and are responsible for the earliest onset cases. These genes encode respectively for parkin and PINK1 proteins, which work together in a mitochondrial quality control process consisting in tagging proteins of damaged mitochondria with ubiquitin molecules. The accumulation of ubiquitinated proteins at the mitochondrial surface triggers the degradation of either the whole damaged mitochondria through mitophagy (Pickles et al, 2018) or the excision of damaged portions through the formation of mitochondrial-derived vesicles (Sugiura et al, 2014). Parkin and PINK1 are also involved in the suppression of mitochondrial antigen presentation (Matheoud et al, 2016) and activation of the STING pathway (Sliter et al, 2018), suggesting a role of inflammation in Parkinson’s disease. Parkin is a cytosolic E3-ubiquitin ligase composed of a N-terminal ubiquitin-like domain (Ubl) linked to a R0RBR module formed by four zinc-binding domains: RING0, RING1, IBR, and RING2 (Figs 1A and EV1). As an RBR-type E3 enzyme, parkin mediates the transfer of ubiquitin from an E2 enzyme onto a cysteine in the RING2 domain, followed by a second transfer onto the substrate protein (Wenzel et al, 2011). X-ray structures of parkin have revealed that it adopts an autoinhibited conformation in basal cell conditions (Riley et al, 2013; Trempe et al, 2013; Wauer & Komander, 2013) in agreement with its previously reported autoinhibition (Chaugule et al, 2011). The active cysteine located on the RING2 domain is partially occluded by its RING0 domain, and the E2-binding site on RING1 is blocked by the parkin Ubl domain and a short α-helix referred to as the repressor element of parkin (REP). In cells, parkin needs to both translocate to mitochondria and undergo a conformational change to release the autoinhibition. Parkin recruitment to mitochondria depends on PINK1, a serine/threonine kinase, which acts as a sensor of mitochondrial defects (Geisler et al, 2010; Matsuda et al, 2010; Narendra et al, 2010; Vives-Bauza et al, 2010). PINK1 accumulates at the surface of damaged mitochondria when mitochondrial protein import is impaired. There, it phosphorylates ubiquitin molecules present at mitochondrial surface. Parkin binds phosphorylated ubiquitin (pUb) with high affinity, which promotes its accumulation onto mitochondria (Kane et al, 2014; Kazlauskaite et al, 2014; Koyano et al, 2014; Ordureau et al, 2014). Parkin is then in turn phosphorylated by PINK1 to activate its ubiquitination activity (Kondapalli et al, 2012; Kazlauskaite et al, 2015; Kumar et al, 2015; Sauve et al, 2015; Wauer et al, 2015a). The most recent x-ray structures of the complex of phosphorylated parkin (pParkin) and pUb have revealed that the phosphorylation of the parkin Ubl domain (pUbl) is responsible for the structural rearrangement within parkin that exposes the active cysteine (Gladkova et al, 2018; Sauve et al, 2018) (Fig EV1). Phosphoserine 65 of pUbl binds a phosphate-binding site on RING0 formed by residues K161, R163, and K211. The relocation of the pUbl domain displaces the catalytic domain RING2 from RING0. The released RING2 domain is free to reposition next to the ubiquitin-charged E2 for the transfer of ubiquitin to the parkin active cysteine and subsequently to target proteins. The addition of new ubiquitin molecules to the mitochondrial outer surface provides more substrates for PINK1, which leads to additional parkin recruitment to mitochondria (Seirafi et al, 2015). This positive feedback mechanism amplifies the signal for mitophagy and explains the observation that parkin activity is required for its recruitment (Lazarou et al, 2013; Ordureau et al, 2014; Shiba-Fukushima et al, 2014). Figure 1. Parkin recruitment and mitophagy without Ubl phosphorylation Schematic representation of parkin highlighting key residues involved in parkin activation and activity. Partial recruitment to mitochondria of parkin mutants without the Ubl-phosphorylation site. Graph shows recruitment of GFP-parkin WT, K211N, C431S, and ΔUbl (deletion of residues 1–76), ΔUbl K211N, S65A, S65A K211N measured in U2OS cells 120 min after depolarization of mitochondria by 20 μM CCCP. Mutation K211N of the RING0 pUbl-binding site decreased recruitment even in the absence of Ubl-phosphorylation. Results are shown from two independent biological replicates with two-way analysis of variance (ANOVA) with Bonferroni post-test. Mitophagy in the absence of Ubl-phosphorylation. Mitophagy was detected by fluorescence-activated cell sorting (FACS) of untreated and CCCP-treated (20 μM for 4 h) U2OS cells containing mitochondrially targeted mKeima (mt-Keima) and transiently expressing GFP-parkin. The K211N mutation was epistatic to the Ubl-phosphorylation and decreased mitophagy to the levels seen in the parkin catalytically dead C431S mutant. For statistical analysis, a one-way ANOVA with Tukey’s post-test was performed on data from three biological replicates. Error bars indicate s.e.m. Lack of complementation between parkin mutants defective in E2-binding and ubiquitin transfer. U2OS mt-Keima cells were transfected with CFP or GFP alone (labeled: -) or as parkin fusion proteins and mitophagy measured by FACS for untreated and CCCP-treated (20 μM for 12 h). For statistical analysis, a one-way ANOVA with Tukey’s post-test was performed on data from three biological replicates. Error bars indicate s.e.m. Data information: *P < 0.05, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Conformational changes in parkin upon phosphorylation or pUb binding Full-length parkin and the R0RBR module have low basal ubiquitin ligase activity due to masking of the RING2 catalytic site (cysteine 431) by the RING0 domain. Parkin is recruited to damaged mitochondria by a high-affinity site on the RING1 domain that binds phospho-ubiquitin (pUb). Upon phosphorylation of the Ubl domain or the binding of a second pUb, parkin becomes activated as the phosphorylated Ubl domain (pUbl) or pUb binds to a site on RING0 releasing and exposing the RING2 catalytic site. Download figure Download PowerPoint Parkin is also regulated through a feedforward (open-cycle) mechanism, which does not require parkin phosphorylation. Multiple studies have shown that deletion of the Ubl domain or loss of serine 65 does not completely abolish parkin recruitment to mitochondria and mitophagy (Shiba-Fukushima et al, 2012; Ordureau et al, 2014; Zhuang et al, 2016; Tang et al, 2017). While unphosphorylated parkin can be activated in vitro by pUb addition, the molecular mechanism has remained unexplored (Kazlauskaite et al, 2014). Here, we confirm the existence of a secondary mechanism for parkin activation that is independent of parkin phosphorylation but dependent on the RING0 pUbl-binding site. We observe that pUb can bind to the pUbl-binding site and, in fact, has higher affinity than the pUbl domain in trans. Experiments with phosphorylated polyUb chains reveal the avidity of the pUb and pUbl-binding sites and mimic the effect of multiple immobilized pUb molecules in proximity to each other on the surface of mitochondria. Finally, experiments with parkin with the Ubl domain replaced by ubiquitin show the chimeric molecule is readily activated by phosphorylation. The feedforward mechanism increases the robustness of the pathway for the clearance of damaged mitochondria and likely represents an early feature in the evolutionary development of the PINK1/parkin pathway. Results Parkin recruitment and mitophagy in cells To verify that parkin in cells could be activated in the absence of Ubl phosphorylation, we monitored the recruitment of different parkin variants (Fig 1A) from cytoplasm to mitochondria upon the addition of mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (Fig 1B). WT parkin was actively recruited with over 80% of cells showing puncta of parkin on mitochondria after 2 h (Appendix Fig S1). The C431S mutant, which has no ligase activity, displayed the lowest recruitment as expected from its incapacity to ubiquitinate mitochondrial proteins and drive the PINK1/parkin feedback cycle. As observed by others, parkin bearing the S65A mutation or deletion of the Ubl domain (∆Ubl parkin) showed partial recruitment even though the mutations prevent parkin phosphorylation. The role of the RING0 pUbl-binding site was evident as recruitment was further reduced by a second mutation (K211N) that disrupts the phosphoserine binding site (Wauer et al, 2015a; Gladkova et al, 2018; Sauve et al, 2018). These results demonstrate a mechanism of parkin activation that is independent of parkin phosphorylation but dependent on the pUbl-binding site on RING0. mt-Keima assays were performed to assess the level of mitophagy in cells expressing parkin mutants. The assay measures mitophagy in cells expressing a mitochondrially targeted fluorophore that shifts its excitation spectrum when mitochondria enter the acidic environment of lysosomes. Addition of the protonophore CCCP to depolarize mitochondria induced mitophagy in cells expressing WT parkin (Fig 1C and Appendix Fig S2). Loss of the parkin active cysteine (C431S) or pUbl-binding site (K211N) abolished the mitophagy response but cells expressing non-phosphorylatable S65A parkin or ∆Ubl parkin showed partial mitophagy. The addition of the K211N mutation to inactivate the RING0 pUbl-binding site eliminated the remaining mitophagic activity. These results demonstrate that an alternative mechanism, involving the pUbl-binding site, can trigger mitophagy of depolarized mitochondria. Parkin does not show complementation between inactivating mutations A recurring model of parkin activity has posited that parkin forms dimers or oligomers to allow the catalytic RING2 domain of one molecule to contact the E2~Ub bound to another molecule (Gundogdu et al, 2021). We previously showed an absence of complementation in autoubiquitination assays (Sauve et al, 2018), but this left open the possibility of trans-active parkin dimers on the surface of mitochondria. To test this, we looked for complementation in the mt-Keima assay (Fig 1D) between the C431S mutant, which is catalytically dead, and the T240R mutant (Fig 1A), which is unable to bind E2 enzymes (Appendix Fig S3A). The mutants were tagged with GFP and CFP, so their expression could be measured and only cells expressing both proteins counted (Appendix Fig S3B and C). The mt-Keima assay showed robust mitophagy in cells expressing WT parkin after 12 h of CCCP treatment. No mitophagy was detected in cells expressing the T240R and C431S mutants individually or together. Due to variations in the basal signal, the absence of complementation can best be seen in the individual experiments where cells expressing both mutants show no more mitophagy than cells expressing them separately with GFP or CFP (Fig 1D and Appendix Fig S3D). These results strongly argue against the existence of trans-active parkin oligomers. pUb binding activates parkin We used two different in vitro assays to test whether pUb binding could activate parkin in the absence of Ubl phosphorylation. The first uses ubiquitin vinyl sulfone (UbVS), which is a chemically reactive derivative that crosslinks to the parkin active-site cysteine (Borodovsky et al, 2001). The formation of the UbVS adduct is a sensitive measure of exposure of the parkin catalytic site and has been widely used to follow parkin activation (Riley et al, 2013; Ordureau et al, 2014; Wauer et al, 2015a). The advantages of the UbVS assay are its simplicity. It only measures accessibility of the parkin active-site cysteine and does not require E1 and E2 enzymes or acyl transfer of ubiquitin to lysine residues. We used the R0RBR construct that is missing the Ubl domain but retains the catalytic RING2 domain and the pUb and pUbl-binding sites on RING1 and RING0. Assays were done with R0RBR parkin purified as a complex with pUb. UbVS assays showed that the addition of pUbl could promote the formation of R0RBR-Ub crosslinks due to the release of the RING2 domain from RING0 (Fig 2A, upper left panel, and Appendix Fig S4, middle and lower panels). The activation of parkin could be more easily observed with the W403A mutation (Fig 2A, upper right panel) that partially derepresses parkin activity by destabilizing the REP helix (Trempe et al, 2013) (Fig 1A). Addition of 50 µM pUbl to the W403 mutant generated more than 50% R0RBR-Ub crosslinks. The high concentration of pUbl required reflects the competition between RING2 and pUbl for binding RING0 and the fact that the RING2 domain is present in the same polypeptide chain at a high local concentration. We used the K211N mutation in the RING0 to confirm the essential role of the pUbl-binding site. No UbVS crosslinks were observed, confirming that the release of RING2 was completely dependent on pUbl-binding RING0 (Fig 2B). Figure 2. Activation of parkin by pUbl or pUb binding Ubiquitin vinyl sulfone (UbVS) assays of RING2 release by addition of pUbl and pUb in trans to R0RBR parkin. Assays were performed with incubation of 2 μM wild-type or W403A R0RBR purified in a 1:1 complex with pUb. Crosslinking was initiated by addition of 10 μM UbVS in the presence of pUbl or pUb∆G76. Inhibition of R0RBR activation by the K211N mutation. UbVS assays of RING2 release by various types of pUb. Autoubiquitination assays of parkin ligase activity induced by pUbl or pUb∆G76 in trans. 2 µM GST-R0RBR in complex with pUb was incubated with 50 nM E1, 2 µM UbcH7, 100 µM ubiquitin, and 4 mM ATP. UbVS assays of RING2 release with R0RBR purified without pUb bound. Autoubiquitination assays of parkin ligase activity with GST-R0RBR without pUb bound. The percentage of unmodified parkin band in each lane is indicated under the gel. Download figure Download PowerPoint We next asked if pUb could similarly release the RING2 domain and generate UbVS crosslinks. The assays used pUb∆G76 (pUb without the C-terminal glycine) for consistency with subsequent autoubiquitination assays where pUb interferes with the E2 discharging (Ordureau et al, 2014; Wauer et al, 2015b). Similar results were obtained with full-length pUb (Fig 2C). Surprisingly, pUb∆G76 was much more efficient than pUbl in producing crosslinks, which suggests that it has a higher affinity for the RING0 pUbl-binding site (Fig 2A, lower left panel). Addition of pUb∆G76 to wild-type R0RBR led to detectable crosslinking at 20 µM and more than 40% at 200 µM. The R0RBR W403A mutant was again more sensitive with 50% crosslinking at 7 µM pUb∆G76 and nearly 100% at higher concentrations (Fig 2A, lower right panel). Assays with the K211N mutant confirmed that RING0 pUbl-site was required for pUb-induced crosslinks (Fig 2B). Only pUb adopting the canonical, major ubiquitin conformation (Wauer et al, 2015b) could bind RING0 pUbl-binding site as shown by the absence of crosslinking in the presence of the pUb mutant TVLN that stabilizes the minor conformation (Gladkova et al, 2017) (Fig 2C). In a second set of assays, we measured autoubiquitination of GST-R0RBR parkin upon the addition of pUbl or pUb (Fig 2D). We used GST-tagged parkin because it displays a higher activity than untagged parkin, most likely due to lysine residues in GST acting as additional sites for ubiquitination. In agreement with the UbVS assays, addition of pUbl in trans activated parkin ligase activity detected by loss of the unmodified GST-R0RBR parkin and the formation of higher molecular weight bands. pUb (Fig 2D, lower panel) was again more efficient than pUbl (Fig 2D, upper panel) in releasing parkin autoinhibition and required 10-fold lower concentrations for activation. To confirm the importance of the RING0 Ubl-binding site for parkin activation, we again used a K211N mutant. No autoubiquitination of K211N GST-R0RBR was observed even at 200 µM pUb (Fig 2D). To compare the efficiency of parkin activation in trans and in cis, we measured the autoubiquitination activity of 2 µM GST-R0RBR/pUb with 2 µM pUbl (Fig 2D, upper panel) and phosphorylated full-length parkin in the presence of pUb (Fig EV2A, left panel). Only 24% of unmodified parkin band was still present in the in cis reaction (Fig EV2A, left panel) compared to 83% for the in trans reaction (Fig 2D, upper panel). This shows that activation in cis is more efficient. To rule out possible artifacts due to the presence of the GST tag, we repeated the in trans assays with R0RBR without a tag and a longer incubation time to compensate for the reduced autoubiquitination activity of untagged parkin. The results with untagged R0RBR (Fig EV2B) showed the same trend than those with GST-parkin (Fig 2D). Parkin was most efficiently activated by pUbl in cis (Fig EV2A, right panel); however, when added in trans, pUb (Fig EV2B, lower panel) was more efficient than pUbl (Fig EV2B, upper panel). Click here to expand this figure. Figure EV2. Activation of parkin by pUbl or pUb binding Autoubiquitination assays of parkin ligase activity of inactive and activated full-length parkin. Assays were performed with incubation of 2 μM of parkin or phosphorylated parkin purified in a 1:1 complex with pUb under the same conditions as autoubiquitination assays with GST-R0RBR/pUb (Fig 2D) and R0RBR/pUb. Autoubiquitination assays of parkin ligase activity induced by pUbl or pUb∆G76 in trans. 2 µM R0RBR in complex with pUb was incubated with 50 nM E1, 2 µM UbcH7, 100 µM ubiquitin, and 4 mM ATP. The percentage of unmodified parkin band in each lane is indicated under the gel. Download figure Download PowerPoint The UbVS and autoubiquitination assays used R0RBR parkin in complex with a stoichiometric amount of pUb bound to the RING1 site. To investigate possible coupling between the RING1 and RING0 sites, we repeated the pUbl titrations without pUb present. The UbVS assay showed a small decrease in the effectiveness of pUbl additions (Fig 2E). W403A R0RBR complexed with pUb required roughly 30 µM pUbl to achieve 50% crosslinking (Fig 2A, upper right panel) while the sample without pUb required 100 µM pUbl (Fig 2E, right panel). Autoubiquitination assays showed a much larger difference. Approximately, 10-fold more pUbl was required to activate GST-R0RBR parkin in the absence of pUb (Fig 2F) than in its presence (Fig 2D, upper panel). Increased local concentration enhances parkin activation by pUb To compare activation by pUb and pUbl in the context of intact parkin, we designed a chimeric molecule in which the Ubl domain has been replaced by ubiquitin (Fig 3A). To prevent pUb binding to the high-affinity site on RING1, we introduced the A320R mutation that disrupts the RING1 site (Wauer et al, 2015a). The chimeric protein could be phosphorylated by PINK1 (Fig 3B) and crosslinked to UbVS as efficiently as wild-type parkin (Fig 3C). The RING0 binding site was essential as the K211N mutation prevented crosslinking. Autoubiquitination assays showed that the chimera and wild-type parkin had identical ubiquitination activity when phosphorylated, and activity was again completely blocked by K211N mutation in the pUbl-binding site (Fig 3D). These results demonstrate that ubiquitin can fully replace the Ubl domain of parkin and, when phosphorylated, binds to RING0 to release the catalytic RING2 domain. Figure 3. Ubiquitination activity of Ub-R0RBR chimera Schematic representations of phosphorylated parkin (pParkin) and the phosphorylated chimera (pUb-R0RBR) with the A320R mutation. Phosphorylation of wild-type (WT) parkin and chimeric Ub-R0RBR by PINK1. Reactions were analyzed on a Phos-tag SDS–PAGE gel to assess the level of phosphorylation. Release of RING2 by Ub-R0RBR chimera. Ubiquitin vinyl sulfone assays (10 μM UbVS) were performed with 3 μM phosphorylated and non-phosphorylated full-length parkin and Ub-R0RBR. Ubiquitination activity of Ub-R0RBR chimera. Autoubiquitination assays of phosphorylated full-length parkin and Ub-R0RBR (3.3 µM with 3 µm UbcH7 and 75 µM S65A ubiquitin) were performed to assess the impact of pUbl substitution by pUb on parkin activity. Download figure Download PowerPoint On damaged mitochondria, PINK1 phosphorylation of ubiquitin molecules likely leads to an elevated local concentration of pUb. Mono-ubiquitin and polyubiquitin chains can be phosphorylated by PINK1 in vitro, and polyphosphorylated ubiquitin chains typically phosphorylated on their terminal Ub molecules have been detected in cells following mitochondrial depolarization (Ordureau et al, 2014; Wauer et al, 2015b; Swatek et al, 2019). Here, we used phosphorylated polyubiquitin chains as a tool to mimic the mitochondrial surface modified with multiple pUb molecules. The existence of two pUb-binding sites on parkin should lead to an increased affinity from cooperation between the sites. Binding of one end of a poly-pUb chain to the high-affinity RING1 site should bring a second pUb molecule in proximity to the low-affinity RING0 site. Fitting of commercially available tetra-pUb chains onto structures of activated parkin showed that they could bridge both pUb-binding sites (Fig 4A). Figure 4. Parkin activation by pUb chains Model of (pUb)4 c" @default.
• W4225289119 created "2022-05-05" @default.
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• W4225289119 title "Structural basis for feedforward control in the PINK1/Parkin pathway" @default.
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