1 site-specific interaction mapping of phosphorylated ubiquitin to

Parkin-Ubiquitin interaction upon phosphorylation

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Site-specific interaction mapping of phosphorylated ubiquitin to uncover Parkin activation

Koji Yamano1,*, Bruno B. Queliconi2, ¶ , Fumika Koyano1, ¶, Yasushi Saeki2, Takatsugu Hirokawa3, Keiji Tanaka2, and Noriyuki Matsuda1,*

1Ubiquitin project, 2Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan 3Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan ¶ These authors equally contributed to this work. * Corresponding authors: Koji Yamano and Noriyuki Matsuda Ubiquitin project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan. Tel: +81-3-5316-3123, Fax: +81-3-5316-3152 Email: [email protected] and [email protected] Keywords: E3 ubiquitin ligase; mitochondria; mitophagy; Parkinson’s disease; PINK1 Background: Phosphorylation of both Parkin and ubiquitin by PINK1 is crucial for Parkin E3 ligase activity. However, the mechanism remains unknown. Results: Site-specific photo-crosslinking identified the phosphorylation-dependent interaction surface between Parkin and ubiquitin. Conclusion: IBR along with RING1 domain of Parkin provides an interaction site for ubiquitin. Significance: A novel binding mechanism with phosphorylated ubiquitin leads to a Parkin conformational change. ABSTRACT Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser65 by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the In-Between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay also showed pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on

the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. Two gene products mutated in autosomal recessive forms of familial Parkinsonism, Parkin and PINK1 (1,2), have been identified as essential proteins for eliminating damaged mitochondria through autophagy machinery in a process called mitophagy (3,4). In healthy mitochondria, a serine/threonine kinase PINK1 is imported to mitochondria in accordance with its N-terminal mitochondrial targeting sequence. After processing by MPP (mitochondrial processing peptidase) and PARL (presenilin-associated rhomboid-like) proteases (5-8), cleaved PINK1 is retrotranslocated to the cytosol and rapidly degraded by the proteasome through an N-end rule pathway, thereby maintaining low PINK1 levels (9). In contrast, when mitochondrial membrane potential is disrupted, PINK1 is stabilized on the outer membrane with its kinase domain exposing cytosolic face (10,11). Another gene product linked to familial Parkinsonism, Parkin, is an E3 ubiquitin ligase that is normally localized throughout the cytosol as an inactivated form (12,13). The role of E3 ubiquitin ligases is to mediate ubiquitin transfer from an E2 ubiquitin-conjugating enzyme to substrate proteins. E3 ligases are classified into three groups based on the protein structure: HECT, RING, and RBR (RING-between-RING). Parkin is a member of the RBR-type ligases. Although RBR-type ligases are typically composed of three conserved structural domains called RING1, IBR (In-Between-RING), and RING2, Parkin has two additional subdomains situated at the N-terminus termed Ubl (Ubiquitin-like) and RING0 and a third

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.671446The latest version is at JBC Papers in Press. Published on August 10, 2015 as Manuscript M115.671446

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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subdomain termed REP (Repressor Element of Parkin) between the IBR and RING2 domains (Fig. 1A). Based on the crystal structure of Parkin, the catalytic Cys431, which forms a thioester intermediate with ubiquitin, and the E2 binding site in the RING1 domain are occluded by the RING0 and REP domains, respectively, indicating that Parkin E3 ligase activity is normally intramolecularly autoinhibited (14-17). Following activation, cytosolic Parkin translocates to depolarized mitochondria (18) where it ubiquitinates various mitochondrial outer membrane proteins (19-21). Recent accumulating evidence has shown that PINK1 stabilized on the outer membrane phosphorylates both the Ubl domain of Parkin and ubiquitin at Ser65 to convert inactivated Parkin to the fully activated form (22-27). Furthermore, phosphorylated Parkin selectively associates with phosphorylated ubiquitin, and PINK1 promotes phosphorylation of poly-ubiquitin chain as well as mono-ubiquitin. Based on these results, a positive feed forward ubiquitination cycle has been proposed to accelerate Parkin translocation onto mitochondria as well as robust ubiquitination of mitochondrial outer membrane proteins (28-30). Therefore, understanding the molecular mechanisms underlying the critical initial step for mitophagy (i.e. how phosphorylated ubiquitin binds to phosphorylated Parkin and how the interaction promotes Parkin E3 ubiquitin ligase activity) is of significant interest. When the C-terminal glycine of ubiquitin is activated through an E1-E2 cascade reaction, Parkin can load the ubiquitin on its catalytic Cys431 via ubiquitin-thioester intermediate (14,24,31,32). However, both Parkin Cys431 and the ubiquitin C-terminal glycine are dispensable for the interaction between phosphorylated Parkin and ubiquitin (27). Therefore, an uncharacterized, phosphorylation-dependent interaction site between Parkin and ubiquitin must exist. Experimental procedures Antibodies The following antibodies were used for immunoblotting: mouse anti-Parkin (Sigma, clone PRK8, P6248), mouse anti-ubiquitin (Santa Cruz Biotechnology, clone P4D1, sc-8017), mouse anti-GST (Santa Cruz Biotechnology, clone B-14, sc-138), and mouse anti-HA (MBL, clone TANA2, M180-3). Rabbit anti-TOMM20 (Santa Cruz Biotechnology, sc-11415) antibody was used for immunostaining. Plasmids For in vivo site-specific crosslinking, GST-rat

Parkin (S65E) and His6-human ubiquitin (S65D) genes were subcloned into the NcoI/NotI and NdeI/XhoI sites of pETDuet-1 vector (Novagen), respectively. To prepare recombinant GST-rat Parkin, GST-TcPINK1 and His6-human ubiquitin, pGEX6P1-ratParkin, pGEX6P1-TcPINK1 (gifts from Jean-François Trempe), and pT7-7/His-Ub plasmids were used. For preparation of BPA-incorporated recombinant GST-rat Parkin, first, the stop codon in pGEX6P1-ratParkin plasmid (originally an amber codon) was changed to TAA (ochre codon) and then the amber codon was introduced at various amino acid positions by primer-based PCR mutagenesis. pEVOL-pBpF (Addgene, Plasmid #31190) was used for expressing the orthogonal pair of the amber suppressor tRNA and its BPA aminoacyl tRNA synthetase in bacterial cells. In vivo site-specific photo-crosslinking Escherichia coli BL21(DE3) cells harboring pEVOL-pBpF and pETDuet/GST-ratParkin (S65E, Xamber, whre x indicates amino acid residues replaced with the amber codon) & His6-human ubiquitin (S65D) were grown in 8 ml of LB medium supplemented with 100 µg/ml ampicillin and 25 µg/ml chloramphenicol at 37°C until an OD600 of ~0.8. The cell culture was then shifted to 16°C before adding 0.1%(w/v) arabinose (Sigma) and 1 mM p-benzoyl-phenylalanine (BPA) (BACHEM). BPA-incorporated GST-rat Parkin (S65E) and His6-human ubiquitin (S65D) were expressed by addition of 50 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 25 µM ZnCl2 for 16 hours at 16°C. The bacterial culture was split in half with one aliquot subjected to UV irradiation (365 nm) using a B-100AP (UVP) for 5 min. The cell pellet after centrifugation was stored -80°C until needed. The frozen cell pellet was solubilized with 500 µl of B-PER Bacterial Protein Extraction Reagent (Thermo Scientific) supplemented with 100 µg/ml lysozyme, 5 units/ml DNAseI, and protease inhibitor cocktail (Roche) for 10 min at room temperature. The supernatants were clarified by centrifugation and mixed with 500 µl of solubilization buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 6M urea, 0.5% (v/v) Triton X-100) and 80 µl of 50%(v/v) equilibrated Ni-NTA agarose (Qiagen)). His6-human ubiquitin (S65D) and its crosslinked products were bound to Ni-NTA by mixing for 15 min at room temperature and then were washed with 1 ml wash buffer (solubilization buffer containing 20 mM imidazole) for 4 times. Proteins were eluted with 40 µl of SDS-PAGE sample buffer. Preparation of recombinant proteins


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GST-rat Parkin, GST-TcPINK1, and His6-human ubiquitin were overexpressed in Escherichia coli BL21(DE3) cells from the aforementioned plasmids. The transformants were grown to OD600 ~0.8 in LB medium supplemented with 100 µg/ml ampicillin at 37°C. GST-rat Parkin and GST-TcPINK1 were induced by addition of 25 µM IPTG for 12 hours at 16°C. For GST-rat Parkin expression, 25 µM ZnCl2 was also added. Bacterial cells were harvested by centrifugation, resuspended in TBS (50 mM Tris-HCl pH7.5, 120 mM NaCl) supplemented with protease inhibitor cocktail (Roche) and 30 µg/ml lysozyme (Sigma), frozen in liquid nitrogen, and stored -20°C. The thawed cell resuspension was sonicated (Advanced-Digital Sonifier, BRANSON), and cell debris and insoluble proteins were removed by centrifugation (12,000 × g, 20 min, 4°C). The soluble fraction was then mixed with Glutathione-Sepharose 4B (GE Healthcare) for 30 min at 4°C. The sepharose was loaded onto a column, washed with TBS buffer, and GST-tagged proteins were eluted with TBS containing 20 mM L-Glutathione reduce (Sigma). L-Glutathione was removed by either dialysis or via a PD Miditrap G-25 (GE Healthcare). Samples were concentrated using Amicon Ultra centrifugal filters (Millipore). GST-rat Parkin (45 µM) was dissolved in TBS supplemented with 1 mM dithiothreitol (DTT) and 10%(w/v) glycerol, whereas GST-TcPINK1 (12 µM) was dissolved in TBS supplemented with 10%(w/v) glycerol. His6-human ubiquitin was induced with 1 mM IPTG at 37°C for 3 hours. The soluble cell fraction prepared as described above was mixed with Ni-NTA, washed with TBS and the His6-human ubiquitin was eluted using 200 mM imidazole. After dialysis and protein concentration, His6-human ubiquitin (750 µM) was dissolved in TBS and stored at -80°C. In vitro kinase reaction with recombinant TcPINK1 Recombinant His6-human ubiquitin was incubated with recombinant GST-TcPINK1 at a molar ratio of 20:1 in kinase buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 10 mM ATP, 50 mM MgCl2, 2 mM DTT) at 32°C for 80 min. Samples were solubilized with SDS-PAGE sample buffer. In the case of Parkin phosphorylation, recombinant GST-rat Parkin was incubated with GST-TcPINK1 at a molar ratio of 2:1 in the presence of recombinant His6-human ubiquitin. Phos-tag PAGE To separate phosphorylated proteins from non-phosphorylated proteins on SDS-PAGE, samples were loaded onto 7.5% or 14% polyacrylamide gels containing 100 µM MnCl2 and 50 µM Phos-tag Acrylamide (Wako). After

electrophoresis, proteins were stained using an Oriole fluorescent gel staining dye (Bio-Rad) or immunoblotted using anti-ubiquitin or anti-Parkin antibodies. In vitro site-specific photo-crosslinking When BPA-incorporated His6-human ubiquitin was used for photo-crosslinking, recombinant GST-TcPINK1, GST-rat Parkin and His6-human ubiquitin were incubated at a molar ratio of 1:2:20. When BPA-incorporated GST-rat Parkin was used for photo-crosslinking, recombinant GST-TcPINK1, GST-rat Parkin and His6-human ubiquitin were used at a molar ratio of 1:2:6. After in vitro phosphorylation, samples were split in two with one aliquot subjected to UV irradiation for 10 min, and the other kept on ice. Proteins were separated by SDS-PAGE and stained with Bio-Safe Coomassie G-250 staining dye (Bio-Rad). The coomassie-stained gels were imaged on an Image Quant LAS 4000 system (GE Healthcare). Image J software was used for quantification of coomassie-staned bands. Modification of Parkin Cys431 by ubiquitin-vinyl sulfone After in vitro phosphorylation with or without GST-TcPINK1, saturating amounts of ubiquitin-vinyl sulfone (Boston Biochem) were added (1:5 molar ratio of GST-rat Parkin and ubiquitin-vinyl sulfone) and the samples were further incubated at 25°C for various time points. The reaction was stopped by adding SDS-PAGE sample buffer. Proteins were subjected to SDS-PAGE followed by immunoblotting with anti-Parkin and anti-HA antibodies. Immunoblotting The appropriate amounts of proteins were separated on 4-12% Bis-Tris SDS-PAGE gels (Invitrogen) using MES or MOPS running buffers (Invitrogen). After transfer, PVDF membranes were blocked and incubated with primary antibodies. Proteins were detected using alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology, sc-2008 or sc-2007) as secondary antibodies and a BCIP-NBT solution kit (Nacalai Tesque). Image J software was used for quantification of protein bands. Size-based gel filtration Gel filtration was carried out on an ÄKTA purifier system (GE Healthcare) using a Superdex 75 10/300 column (GE Healthcare). After in vitro phosphorylation with or without GST-TcPINK1, samples were loaded onto a Superdex 75 column equilibrated with TBS buffer at a flow rate of 0.4


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ml/min, and 1ml fractions were collected. Proteins were precipitated with trichloroacetic acid, solubilized with SDS-PAGE sample buffer, and analyzed by CBB staining or immunoblotting. A gel filtration LMW calibration kit (GE Healthcare Cat# 28-4038-41) was used for protein weight markers. Fluoppi assay HeLa cells were cultured in Dulbecco’s modified Eagle’s (DMEM, Sigma) medium supplemented with 10%(v/v) fetal bovine serum (Gibco), non-essential amino acids (Gibco), sodium pyruvate (Gibco), and Pen Strep glutamine (Gibco) at 37°C in a 5% CO2 incubator. Plasmids used in the Fluoppi analysis were constructed as follows. Alanine mutations were introduced in the C-terminal diglycine of ubiquitin (G75A and G76A) to prevent a conjugation-derived pseudo-positive signal. The phosphomimetic ubiquitin (S65D) gene was inserted into phAG-MCL plasmid (Amalgaam) to express hAG (homo tetramer Azami-GFP)-tagged ubiquitin. The catalytic site of human Parkin (Cys431) was mutated to alanine (C431A) to prevent ubiquitin conjugation. The phosphomimetic Parkin (S65E) gene was inserted into pAsh-MCL plasmid (Amalgaam) to express Ash (hono-oligomerized protein assembly helper)-tagged Parkin. To generate truncation and pathogenic mutation plasmids, primer-based PCR mutagenesis was used. These plasmids were transfected using FuGENE 6 (Promega) according to the manufacturer’s instructions. Images of the hAG-Ub (S65D) derived foci were obtained using a confocal microscope (LSM710, Carl Zeiss). For image analysis, ZEN2011 (Carl Zeiss) and Photoshop (Adobe) were used. Immunostaining Parkin-transfected HeLa cells grown on 35mm glass bottom dishes were treated with 10 µM valinomycin (Sigma) for 2.5 hrs. The cells were fixed with 4% paraformaldehyde in PBS for 25 minutes at room temperature and permeabilized with 0.15%(v/v) Triton X-100 in PBS for 15 minutes. After blocking, the fixed cells were incubated with anti-TOMM20 antibody and then with Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes). The images of the cells were obtained using a LSM710 confocal microscope. Mass spectrometry and quantification of peptides GST-rat Parkin (WT) was crosslinked to His-Ub (I36BPA) after incubation with GST-TcPINK1. GST-rat Parkin (S65E) was crosslinked to His-Ub (S65D, R42BPA) or His-Ub (S65D, R72BPA). The

appropriate amounts of the crosslinked samples, as well as GST-Parkin (WT and S65E) alone, were subjected to SDS-PAGE followed by CBB staining. CBB-stained bands were gel excised and destained using 50 mM ammonium bicarbonate (AMBC)/30%(v/v) acetonitrile (ACN) with agitation for 1 hour. The gel pieces were further washed with 50 mM AMBC/50%(v/v) ACN for 1 hour and then reduced with 10 mM DTT in 50 mM AMBC buffer for 1 hour at 56°C. After washing with 50 mM AMBC buffer, cysteine residues were alkylated with 55 mM iodoacetamide for 45 min at room temperature. Samples were washed once with 50 mM AMBC buffer, twice with 50 mM AMBC/50%(v/v) ACN, and then dehydrated with 100% ACN. Proteins were subjected to in-gel trypsin digestion using 20 ng/µl Trypsin Gold (Promega) in 50 mM AMBC/5%(v/v) ACN buffer at 37°C overnight. The resulting peptides were solubilized in 0.1% trifluoroacetic acid and analyzed using a nanoflow ultra-HPLC (Easy nLC, Thermo Scientific) coupled to a Q Exactive mass spectrometer (Thermo Scientific). The mobile phases were 0.1% formic acid in water (solvent A) and 0.1% formic acid in 100% ACN (solvent B). Peptides were directly loaded onto a C18 analytical column (Reprosil-Pur 3µm, 75 mm id ×12 cm packed tip column, Nikkyo Technos) with a flow rate of 300 nl/min. For ionization, 1.8 kV of liquid junction voltage and a capillary temperature of 250°C were used. For peptide identification, MS spectra were analyzed using Protein Discoverer version 1.3 (Thermo Scientific). The fragmentation spectra were searched using amino acid sequences of GST-rat Parkin and His6-human ubiquitin. Methionine oxidation and cysteine carbamidomethylation were chosen as static modifications for database searching. For quantification, raw files were processed using PinPoint version 1.3 (Thermo Scientific). Computational modeling of fully activated Parkin with phosphorylated ubiquitin The proposed model for fully activated Parkin with phosphorylated ubiquitin was constructed using the protein-protein docking and molecular dynamics procedure as follows. We first simulated protein-protein docking of phosphorylated ubiquitin (Protein Data Bank ID code: 4wzp) with an inactivated Parkin structure (Protein Data Bank ID code: 4k95). ClusPro version 2.0 (33) was used to generate docked conformations with the lowest docking energies and clustering properties. Phosphorylated Ser65 in phosphorylated ubiquitin was computationally mutated to glutamate because the ClusPro program only supports native amino acids. During the protein-protein docking procedure,


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photo-crosslinking data were utilized as constraints for residues (I36, R42 and R72 of phosphorylated ubiquitin and Y315-R334 and V350-R366 of Parkin) involved in complex formation of the protein-protein interaction interface. The best model in the first step was selected using criteria consisting of: docking energy, number of cluster members and compatibility with the BPA photo-crosslinking experimental results. In the second step, the best model in the first step was subjected to 100 ns molecular dynamics-based energy minimization using Desmond version 3.8 (34); this generated the final model. To prepare the protein structure for molecular dynamics, the missing loop regions of Parkin were constructed using Prime (Schrödinger, LLC). Because the missing loop region from P73 to K140 is long and predicted to be disordered, we truncated the loop by 10 residues leading out of the Ubl domain and 10 residues leading into RING0 domain. Ser65 in the Parkin Ubl domain was also computationally mutated to phosphorylated serine to generate activated Parkin. The OPLS2005 force field was used for simulations. Initial model structures were placed into TIP3P water molecules solvated with 0.15 M NaCl. After minimization and relaxation of the model, the production MD phase was performed for 100 ns in an isothermal-isobaric (NPT) ensemble at 300 K and 1 bar using Langevin dynamics. Long-range electrostatic interactions were computed using the Smooth Particle Mesh Ewald method. All system setups were performed using Maestro (Schrödinger, LLC). All molecular figures were generated using PyMOL (Schrödinger, LLC). RESULTS RING0-RING1-IBR of Parkin is important for the interaction with ubiquitin.

To identify the minimal Parkin domains required for the interaction with phosphorylated ubiquitin, we applied the Fluoppi (fluorescent-based technology detecting protein-protein interaction) technique in which protein interactions are detected as foci in mammalian cells. We previously reported that the Ser65 phosphomimetic pair of Parkin and ubiquitin, but not the wild-type (WT) pair, formed Fluoppi-mediated foci (27). We first confirmed that Ash-tagged phosphomimetic Parkin (Ash-Parkin(S65E)) and hAG-tagged phosphomimetic ubiquitin (hAG-Ub(S65D)) formed foci in HeLa cells (Fig. 1B). When Ash-Parkin(S65E) with Ubl, REP, or RING2 domain deletions was expressed with hAG-Ub(S65D), foci formed with similar efficiency to full-length Ash-Parkin (Fig. 1B and C)

suggesting that these domains are not required for the interaction with ubiquitin. In contrast, deletion of the RING0, IBR or RING1 domains impeded foci formation (Fig. 1B). Next, we examined if pathogenic mutations in the RING0-RING1-IBR region affected the physical interaction between Parkin and ubiquitin. Foci were apparent in cells co-expressing hAG-Ub and Ash-Parkin with R234Q, R256C, N273S, D280N, G328E, T351P or R334C mutations (Fig. 1B and C), whereas R275W mutation reduced numbers of foci. In sharp contrast, L283P, G284R, and C352G mutations inhibited foci formation (Fig. 1B and C). Because Cys352 participates in zinc coordination, the C352G mutation likely disrupts the conformational integrity of the IBR domain. Orientation of the L283 and G284 side chains toward the exterior of the structure suggests that these residues may be directly involved in ubiquitin binding. Taken together, these results indicate that Parkin RING0-RING1-IBR region is important for interacting with phosphorylated ubiquitin and potentially functions to provide a binding surface or to induce conformational flexibility to facilitate stable interactions with phosphorylated ubiquitin. Identification of the Parkin-ubiquitin interaction surface via site-specific photo-crosslinking

To clarify the interaction site between Parkin and ubiquitin in more detail, we applied a site-specific photo-crosslinking technique (35). This technique enables us to introduce a photo-reactive crosslinker, p-benzoyl-phenylalanine (BPA), at desired amino acid positions within a target protein in bacterial cells by using an orthogonal pair of amber suppressor tRNA and its cognate aminoacyl-tRNA synthetase specific for BPA (RpaRS) (36). Subsequent UV irradiation results in covalent bonding of BPA with nearby proteins. Therefore, BPA photo-crosslinking can capture protein-protein interactions at a spatial resolution of amino acid level even if the interaction is weak or transient. A phosphomimetic GST-rat Parkin (GST-rat Parkin (S65E)) gene with an amber codon in a desired position was inserted into pETDuet vector together with a phosphomimetic His6-tagged ubiquitin (His-Ub (S65D)) gene. The plasmid was then introduced into bacterial cells that has the amber suppressor tRNA and BpaRS under an arabinose-inducible promoter. The GST-rat Parkin (S65E, K220amb) fragment, which has an amber codon at K220 position, was converted to the full-length form only when both arabinose and BPA were added to culture media, indicating that BPA was precisely incorporated into the amber codon position (Fig. 2A). Since Fluoppi analysis indicated that the RING0-RING1-IBR domains are important


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for ubiquitin binding, we replaced various amino acids in this region with BPA. After UV irradiation, bacterial cells were subjected to immunobloting, which revealed that when BPA was introduced at residues 266, 282, 327, 329, 344, 355, 356 and 357 in GST-rat Parkin (S65E), bands migrating at ~80 kDa were detected in a UV dependent manner with anti-Parkin antibody (Fig. 2B). The absence of the crosslinked bands in cells lacking exogenous ubiquitin expression confirmed that the crosslinked products were ubiquitin adducts (Fig. 2C). Despite relatively weak intensities, quantification of the crosslinked products indicated that BPA at residues 220, 222, 224, 269, 270, 279, 305, 320, 342, and 347 was also preferentially crosslinked to His-Ub (Fig. 2D). Mapping the ubiquitin-interacting residues onto the inactivated Parkin structure revealed that they segregated across several regions (Fig. 2E). The amino acid positions M327, G329, E344, G355, N356 and G357, whose replacement with BPA generated robust crosslinked products, are clustered within a region on the edge of the IBR domain that is located on the opposite side of the Ubl domain. Other crosslinked residues (score > 0.5), L266 and Q282, are on the α-helix of RING1 domain that directly interacts with Ile44 of the Ubl domain and on the small β-hairpin loop in the RING1 domain, respectively. Weak crosslinked residues (score between 0.25-0.5) such as K220, T222, and V224 are in the RING0 domain.

Next, we tested if these crosslinked products are PINK1 phosphorylation dependent. For this purpose, we performed in vitro photo-crosslinking using recombinant GST-rat Parkin harboring BPA at the desired positions in conjunction with recombinant GST-tagged Tribolium castaneum PINK1 (GST-TcPINK1) (37) and recombinant His-Ub (Fig. 3A). GST-rat Parkin and His-Ub were subjected to in vitro kinase reaction with or without GST-TcPINK1 prior to gel filtration analysis. We confirmed that PINK1 efficiently phosphorylates both Parkin and ubiquitin in vitro (Fig. 3B, IB: Parkin and IB: Ub) as reported previously (22,26). Furthermore, results from the gel filtration analysis indicated that a stable complex only formed when both Parkin and ubiquitin were phosphorylated (Fig. 3B and C). This result is consistent with previous isothermal calorimetry methods which found that the phosphorylated Parkin and ubiquitin pair bound each other tightly with a calculated Kd ~17 nM (29). We then performed in vitro photo-crosslinking using GST-rat Parkin with BPA incorporated at 218, 222, 224, 266, 269, 327, 329, 355, or 357 (Fig. 3D). BPA at residues 327, 329, 355, and 357 in the IBR domain effectively crosslinked with His-Ub in a PINK1 phosphorylation dependent manner. BPA at

residues 222 and 224 also crosslinked with His-Ub, although less efficiently (Fig. 3D and E). On the other hand, L266, which exhibited significant crosslinking capability in bacterial cells (Fig. 2B and D), did not generate a strong crosslinked signal (Fig. 3D and E). In summary, crosslinking occurs in a PINK1 dependent manner, and the crosslinking efficiencies in bacterial cells and in vitro are compatible except for L266.

Next, to determine which region of ubiquitin interacts with Parkin, we prepared recombinant BPA-incorporated ubiquitin (Fig. 4A) and performed in vitro photo-crosslinking. We unexpectedly found that His-Ub containing BPA at residues 42, 45, 44, 49, 70 and 72 was not efficiently (< 20%) phosphorylated by GST-TcPINK1 (Fig. 4B and C). These residues are located adjacent to the β-sheet that is centered at Ile44 (Fig. 4D). Because the BPA side chain is bulky, we assume that steric hindrance blocks the phosphorylation by PINK1. Although further analysis is required, the ubiquitin I44 patch (38) may be important for association with PINK1 prior to phosphorylation. We next sought to further define the ubiquitin interaction surface with Parkin. When the position of BPA was varied, crosslinking to Parkin was the most evident at residue 36 with 70% crosslinking efficiency (Fig. 5A and B). In addition to this, BPA introduction into other ubiquitin residues also resulted in crosslinking to Parkin (Fig. 5A and B). In the case of the amino acid residues where the introduction of BPA inhibited Ser65 phosphorylation (Fig 4C), we substituted the phosphomimetic pair of GST-Parkin and His-Ub (Fig. 5C and D). As the crosslinked efficiency of His-Ub I36BPA (and also R54BPA) to Parkin was comparable between phosphorylated and phosphomimetic forms, we directly compared the crosslinking efficiency between the results generated in Fig. 5B and 5D. Mapping the Parkin-interacting residues onto a phosphorylated ubiquitin structure revealed that K6, T7, L8, G10, K11, I36, R42, and R72 cluster to form the surface that preferentially interacts with Parkin (Fig. 5E). However, BPA at residues 16 and 63, which are located on the other side of ubiquitin was also efficiently crosslinked (Fig. 5E). Site-specific crosslinking combined with mass spectrometry identifies contact points between Parkin and ubiquitin.

In general, the carbonyl oxygen of the benzophenone group of BPA reacts with any carbon-hydrogen bond of a nearby protein, which means that it is difficult to differentiate the crosslinked region of the partner protein at amino acid level. However, we assumed that, when


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compared to non-crosslinked Parkin, a trypsin-treated peptide of Parkin that directly crosslinked to BPA would be shifted. To test this hypothesis, we utilized quantitative mass spectrometry (Fig. 6A). The His-Ub containing BPA at residues 36, 42 and 72 was crosslinked to GST-Parkin and following SDS-PAGE separation, the resulting crosslinked bands were subjected to in-gel trypsin digestion followed by mass spectrometry analysis. Compared to peptides derived from GST (198-218 aa) and Parkin (7-27 aa), the amount of the peptide corresponding to 350-366 aa of Parkin in the His-Ub (I36BPA) crosslinking band was significantly reduced than that of non-crosslinked GST-Parkin. Moreover, the amount of the Parkin 315-334 aa peptide in the His-Ub (R42BPA) or His-Ub (R72BPA) crosslinked products was also reduced compared to the control non-crosslinked GST-Parkin (S65E). A ubiquitin-derived peptide (12-27 aa) was present only in the crosslinked samples, indicating that these bands contained ubiquitin. These results indicate that the spatial coordinates of ubiquitin I36 are in close proximity to the IBR 350-366 aa region of Parkin, and that ubiquitin R42 and R72 are in close proximity to 315-334 aa, which corresponds to the RING1-IBR junction of Parkin. Of note, BPA introduced into these peptides, at 355, 356, 357, 327, and 329 were crosslinked to ubiquitin with high efficiency (Fig. 2D and 3E). Model of interaction between phosphorylated Parkin and phosphorylated ubiquitin

Ubiquitin-vinyl sulfone is a ubiquitin-derived probe with an electrophilic C-terminal end that tergets the active-site cysteine (Cys431) of Parkin. Ubiquitin-vinyl sulfone can thus be used as a tool to monitor Cys431 accessibility. Following binding to phosphorylated ubiquitin, the Parkin catalytic Cys431, which is normally occluded by the RING0 domain, becomes exposed as demonstrated by Ub-vinyl sulfone accessibility (Fig. 6B) (14,24,39,40). This result strongly suggests that Parkin structural remodeling occurs after binding to phosphorylated ubiquitin. We finally constructed superposition model of Parkin and ubiquitin binding by computational modeling. Based on the crosslinking experiments that were coupled to mass spectrometry, we constructed the first model in which ubiquitin I36, R42 and R72 residues are in close proximity to the IBR and RING 1 domains (Fig. 6C). Interestingly, computational simulations suggest that the ubiquitin Ser65 phosphorylation site oriented toward the RING1 domain is captured by the positive-charged residues, K151 and R305 (Fig. 6D and supplemental movie). Further computational

simulations also showed that a separate positive-charged region composed of R170 (and K220) comes into close proximity to the negative-charged surface (E16 and E18) of ubiquitin (Fig. 6D and supplemental movie). The resulting electrostatic interactions drive pushing of the IBR domain with phosphorylated ubiquitin to the RING0 domain, which in turn triggers a conformational change in REP that removes the occlusion blocking as well as bringing the E2 binding site and catalytic Cys431 residue into close proximity (Fig. 6D).

Computational modeling predicted that the ubiquitin I44 patch interacts with the Parkin RING1 domain α-helix (309-326 aa) via hydrophobic-hydrophobic interactions. To test this prediction, we used Fluoppi to monitor Parkin-ubiquitin complex formation following disruption of the hydrophobic-hydrophobic interactions by arginine replacements of A320 or V324 in Parkin or alanine substitution of I44 in ubiquitin. All three mutations completely blocked foci formation (Fig. 7A and B). We also mutated K151 and R305, which are predicted to be critical for coordination with the phosphorylated Ser65. Although the single alanine mutations (K151A and R305A) did not impair the foci formation, the K151A/R305A double mutation did reduce foci formation (Fig. 7A and B). Similar reduction in foci formation in cells expressing a R170E/K220E double mutant suggests that R170 and K220 in Parkin also contribute to interactions with ubiquitin (Fig. 7A and B). We also performed a Parkin translocation assay using these Parkin mutants. While YFP-Parkin wild-type (WT) and the R170E/K220E mutant were efficiently recruited to TOMM20-labeled mitochondria within 2.5 hrs of valinomycin treatment, mitochondrial translocation was completely impeded by the Parkin K151A/R305A, A320R and V324R mutations (Fig. 7C and D). DISCUSSION Numerous studies have shown that PINK1-mediated phosphorylation of both Parkin and ubiquitin at Ser65 is required for efficient Parkin recruitment to damaged mitochondria. More recently, Parkin interactions with phosphorylated ubiquitin, which activate the latent E3 ligase activity of Parkin, have been shown to also be essential to the process. In the current study, we used two independent and unique experimental approaches, Fluoppi and site-specific photo-crosslinking, to elucidate the mechanism by which phosphorylated ubiquitin interacts with Parkin. The Fluoppi-based assay showed that the RING0-RING1-IBR domain of Parkin is essential


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for binding with phosphomimetic ubiquitin (Fig. 1), and pathogenic mutations of this domain, such as L283P, G284R, and C352G, impede those interactions (Fig. 1). Although no ubiquitin cross-linked products were observed following BPA incorporation at Parkin L283 and G284, incorporation at Q282, which is close to L283 and G284, efficiently crosslinked to phosphomimetic ubiquitin (Fig. 2B and 2D). This suggests that L283P and G284R mutations affect ubiquitin binding directly. However, we cannot rule out the possibility that the L283P and/or G283R mutations restrict molecular motion, thereby affecting the flexibility of the small β-hairpin loop comprising V278-S286 aa. Another pathogenic mutation, R275W, also reduced foci formation (Fig. 1). When this mutant was expressed in SH-SY5Y neuroblastoma cells, it was found in the Triton X-100 insoluble fraction (41), suggesting that the mutation affects the overall structural folding and stability of Parkin. The Fluoppi foci in Parkin R275W expressing cells, which were observed as clusters near the perinuclear region rather than spherical dots throughout the cytosol (Fig. 1B), are consistent with a structurally impaired Parkin as suggested above. Further site-specific photo-crosslinking analyses allowed us to generate a map of the interactions linking the phosphorylated forms of Parkin and ubiquitin and to construct a computational model of the Parkin-ubiquitin complex. Based on the inactivated human Parkin crystal structure (16), K161, R163, and K211 coordinate a sulphate ion structure, suggesting that they are viable sites for binding the phosphorylated serine of ubiquitin. However, our computational modeling predicts that Parkin K151 and H302 residues are responsible for capturing the phosphorylated serine of ubiquitin (Fig. 6D). Using site-directed mutagenesis, we confirmed that K151/H302 are directly involved in binding phosphorylated ubiquitin as well as efficient translocation of Parkin to damaged mitochondria (Fig. 7). On the other hand, Parkin was efficiently crosslinked following incorporation of BPA at R42 in non-phosphorylated ubiquitin (Fig. 4C). Consequently, interactions between the Parkin RING1 domain α-helix (309-326 aa) and the I44 hydrophobic patch of ubiquitin are phosphorylation independent. Given that BPA crosslinking can capture transient protein-protein interactions, our data show that Parkin and ubiquitin utilize at least two different interaction sites. Although interactions between K151/R305 of Parkin and phosphorylated Ser65 of ubiquitin stabilize the Parkin-ubiquitin complex, it is the interactions between the RING1 domain α-helix (309-326 aa) and the ubiquitin I44 hydrophobic patch that likely

serve as the initial and most essential contact site. Our mutational analyses in which the Parkin A320R and V324R mutations and the ubiquitin I44A mutation completely impair complex formation support this conclusion.

During the manuscript revision process, two independent studies examining the interactions between Parkin and phosphorylated ubiquitin were published. The Muqit group identified Parkin K151/H302 residues as critical residues for binding phosphorylated ubiquitin (42). Of note, two of the four Parkin residues (K151, H302, R305, Q316) that form a phosphate binding pocket were identified in our computational modeling approach. In addition, the Komander group revealed the crystal structure of Pediculus humanus corporis Parkin complexed with phosphorylated ubiquitin (43). To our surprise, both the overall structure and interaction sites at the amino-acid level are very similar to those predicted by our modeling.

In this study, we elucidated a novel binding mechanism that uses the RING1-IBR domain to promote Parkin interactions with phosphorylated ubiquitin, and demonstrate that site-specific crosslinking coupled with mass spectrometry and computational molecular modeling can serve as a powerful tool to provide insight into a dynamic protein conformational change. Acknowledgements We thank Jean-François Trempe and Kalle Gehring for pGEX6P1-ratParkin and pGEX6P1-TcPINK1 plasmids. This work was supported by JSPS PRESTO, KAKENHI Grant Number 23687018, MEXT KAKENHI Grant Numbers 24111557 and 25112522, and the Tomizawa Jun-ichi and Keiko Fund for Young Scientist (to N.M.); by MEXT KAKENHI Grant Numbers 15K19037 (to F.K.); by MEXT KAKENHI Grant Numbers 24112008 (to Y.S.); by JSPS KAKENHI Grant Number 21000012 (to K.T.); by Platform for Drug Discovery, Informatics, and Structural Life Science from MEXT (to T.H.); and by the Takeda Science Foundation (to K.T. and N.M.). Conflict of interest The authors declare that they have no conflict of interest. Author contributions K.Y. conceived and designed the main body of experiments. K.Y., B.B.Q., and F.K. performed experiments. Y.S. contributed to the mass spectrometry experiments and analysis. T.H. contributed to the computational molecular modeling. K.Y., K.T., and N.M. interpreted data


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Figure Legends FIGURE 1. Fluoppi assay demonstrating an interaction between phosphomimetic Parkin and phosphomimetic ubiquitin. (A) Primary structure and domains of human and rat Parkin. (B) Ash-Parkin (S65E), and its truncated and pathogenic mutants were expressed with hAG-Ub (S65D) in HeLa cells. Foci of the hAG-Ub (S65D) were observed using confocal microscopy. Scale bars, 20 µm. (C) Quantification of foci formation in (B). The percentages of cells forming foci are shown. The error bars represent ± SD from three independent replicates. Over 100 cells were counted in each of three replicate wells. FIGURE 2. Site-specific photo-crosslinking using BPA-incorporated Parkin in bacterial cells. (A) Bacterial cells harboring the plasmid encoding His-Ub (S65D) and GST-rat Parkin (S65E) with no amber mutations (none) or an amber mutation at position K220 (K220) were cultured under the indicated conditions. The total cell lysates were analyzed by immunoblotting with anti-GST antibody. Asterisks, double asterisks, and triple asterisks represent N-terminal truncated GST-rat Parkin with BPA incorporated at K220, a GST-rat Parkin fragment truncated at K220, and an N-terminal truncated GST-rat Parkin fragment truncated at K220, respectively. (B) GST-rat Parkin (S65E) with BPA at the indicated amino acid positions and His-Ub (S65D) were expressed in bacterial cells. The cell culture was split into two with one aliquot subjected


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to UV irradiation. GST-rat Parkin (S65E) and its crosslinked products were immunoblotted using anti-Parkin antibody. His-Ub (S65D) was detected by CBB staining. Arrowheads indicate crosslinked products of GST-rat Parkin (S65E) and His-Ub (S65D). Asterisks denote GST-rat Parkin (S65E) translated from an ATG codon other than the first one. w/o denotes GST-rat Parkin (S65E) without BPA incorporation. Molecular mass markers are shown to the right as kDa. (C) GST-rat Parkin (S65E) incorporated BPA at the indicated amino acid positions were expressed with or without His-Ub (S65D) in bacterial cells. The cell cultures were subjected to UV irradiation. GST-rat Parkin (S65E) and its crosslinked products were analyzed by immunoblotting with anti-Parkin antibody. His-Ub (S65D) was detected by CBB staining. Arrowheads indicate crosslinked products of GST-rat Parkin (S65E) and His-Ub (S65D). Asterisks denote GST-rat Parkin (S65E) translated from an ATG codon other than the first one. (D) The crosslinked products in (B) were quantified. The amounts of crosslinked products (Arrowheads in B) relative to those of GST-rat Parkin without UV irradiation are indicated. (E) The relative amounts of crosslinked products with His-Ub (S65D) in (D) were mapped onto the corresponding amino acid residues in the rat Parkin structure (PDB ID: 4K95) via white-blue gradation. The RING0-RING1-IBR domains are shown as molecular surface projections. Ser65 residue is shown in green. FIGURE 3. In vitro site-specific photo-crosslinking using BPA-incorporated Parkin. (A) Recombinant His-Ub, GST-TcPINK1, and BPA-incorporated GST-rat Parkin were analyzed by SDS-PAGE and CBB staining. w/o denotes GST-rat Parkin without BPA incorporation. Truncated Parkin represents GST-rat Parkin fragments in which translation was terminated at the amber codon without BPA incorporation. (B) After in vitro phosphorylation with or without recombinant GST-TcPINK1, the samples containing recombinant GST-rat Parkin and His-Ub were size fractionated via Superdex 75 gel filtration. Fractions #7-15 were analyzed by SDS-PAGE and CBB staining (upper), or Phos-tag PAGE and immunoblotting with anti-Parkin (IB: Parkin) and anti-ubiquitin (IB: Ub) antibodies (lower). (C) The amount of ubiquitin in each fraction in (B) was quantified. Total ubiquitin in Fractions 7-15 was set to 100%. (D) The indicated recombinant BPA-incorporated GST-rat Parkin and His-Ub were phosphorylated in vitro with or without GST-TcPINK1. The samples were subsequently split into two with one aliquot UV-irradiated. Proteins were detected by CBB staining. Arrowheads indicate crosslinked products of GST-rat Parkin and His-Ub. (E) The crosslinked products in (D) were quantified. The amounts of crosslinked GST-rat Parkin with His-Ub relative to those of GST-rat Parkin without UV irradiation are indicated. FIGURE 4. Phosphorylation of BPA-incorporated ubiquitin by recombinant TcPINK1. (A) CBB staining of recombinant His-Ub with BPA incorporation at the indicated amino acid positions. w/o means His-Ub without BPA incorporation. (B) The indicated BPA-incorporated His-Ub was phosphorylated in vitro with or without GST-TcPINK1. The samples were subjected to Phos-tag PAGE followed by Oriole staining. (C) The amount of phosphorylated His-Ub relative to that of His-Ub in a reaction without GST-TcPINK1 in (B) was quantified. (D) Amino acid residues where BPA replacement inhibited ubiquitin Ser65 phosphorylation by GST-TcPINK1 (< 20% as in (C)) are shown in blue on the surface model of the ubiquitin structure (PDB ID: 1ubq). Ser65 residue is shown in green. FIGURE 5. In vitro site-specific photo-crosslinking using BPA-incorporated ubiquitin. (A) Recombinant His-Ub incorporated BPA at the indicated amino acid positions and GST-rat Parkin were incubated with or without GST-TcPINK1. The samples were split into two with one aliquot subjected to UV irradiation. Proteins were detected by CBB staining. Arrowheads indicate crosslinked products of GST-rat Parkin and His-Ub. w/o denotes His-Ub without BPA incorporation. (B) The crosslinked products in (A) were quantified as a ratio of the amount of crosslinked GST-rat Parkin to that of GST-rat Parkin without UV irradiation. (C) Recombinant His-Ub (WT or S65D) incorporated BPA at the indicated amino acid positions was incubated with GST-rat Parkin (WT or S65E) for 10 min at room temperature. The samples were split into


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two with one aliquot subjected to UV irradiation. Proteins were detected by CBB staining. Arrowheads indicate crosslinked products of GST-rat Parkin and His-Ub. (D) The crosslinked products in (C) were quantified as a ratio of the amount of crosslinked GST-rat Parkin to that of GST-rat Parkin without UV irradiation. (E) The amounts of GST-rat Parkin crosslinked products with His-Ub in the presence of GST-TcPINK1 in (B) and those using a phosphomimetic pair of GST-rat Parkin and His-Ub in (D) were mapped onto the corresponding amino acid residues of the phosphorylated ubiquitin structure (PDB ID: 4wzp) via white-blue gradation. Phosphorylated Ser65 is shown in green. FIGURE 6. Contact surface of Parkin with phosphorylated ubiquitin revealed by site-specific crosslinking coupled to mass spectrometry. (A) Recombinant GST-rat Parkin and His-Ub (I36BPA) were incubated with GST-TcPINK1 followed by UV-irradiation. Recombinant His-Ub (S65D, R42BPA) and His-Ub (S65D, R72BPA) were UV-irradiated with GST-rat Parkin (S65E). The above crosslinked samples and non-crosslinked GST-rat Parkin (WT and S65E) were separated by SDS-PAGE. The regions of the interest (red boxes) were gel excised, alkylated with iodoacetamide, and subjected to in-gel trypsin digestion. The samples were analyzed by quantitative mass spectrometry. The peak area was calculated from the chromatogram corresponding to the indicated peptide. (B) Recombinant GST-rat Parkin and His-Ub were phosphorylated in vitro with or without GST-TcPINK1. HA-tagged ubiquitin-vinyl sulfone (Ub-VS) were then added and incubated at 25°C for the indicated times. The proteins were immunoblotted using anti-Parkin (upper) and anti-HA (middle) antibodies. The amounts of GST-Parkin conjugated to Ub-VS were quantified (lower). (C) The docking structure of phosphorylated ubiquitin to the inactivated Parkin was generated based on the experimental data as in (A). I36 of ubiquitin and its crosslinked region of Parkin were shown in orange. R42 and R72 of ubiquitin and their crosslinked region were shown in blue. (D) Computational model of activated Parkin with phosphorylated ubiquitin. E18 and phosphorylated S65 of ubiquitn, and S65, K151, R170, R305 and C431 of Parkin were shown in ball & stick model. See the experimental procedures for the detailed method. FIGURE 7. Parkin mutants that have a defect of binding with phosphorylated ubiquitin. (A) S65E phosphomimetic Ash-Parkin (referred to as WT) and its mutants were expressed with S65D phosphomimetic hAG-Ub (referred to as WT) and its I44A mutant in HeLa cells as the indicated pair in (B). Foci of the hAG-Ub (S65D) were observed using confocal microscopy. Scale bars, 20 µm. (B) Quantification of foci formation in (A). The percentages of cells forming foci are shown. The error bars represent ± SD from three independent replicates. Over 100 cells were counted in each of three replicate wells. (C) The indicated YFP-human Parkin mutants were transiently expressed in HeLa cells. Cells were treated with valinomycin for 2.5 hrs and subjected to immunostaining with anti-TOMM20 antibody. Scale bars, 20 µm. (D) Quantification of Parkin translocation in (C). The percentages of cells having YFP-Parkin on mitochondria are shown. The error bars represent ± SD from three independent replicates. Over 100 cells were counted in each of three replicate wells.


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Ubl RING0 RING1 IBR REP RING2

1 76 141 225 327 378 410 465 (aa)

Parkin:

A

B

Yamano K. et al. Fig1

ΔUbl ΔRING0 ΔRING1 ΔIBR ΔREP ΔRING2Full-length R234Q

R256C N273S R275W D280N L283P G284R G328E R334C

T351P C352G

C

0

20

40

60

80

100

% o

f cel

ls h

avin

g hA

G-U

b fo

ci

Ash-Parkin:(S65E)

ΔUblΔRING0

ΔRING1

ΔRING2

ΔIBRΔREP

Full-length

R234QR256C

N273SR275W

D280NL283P

G284RG328E

T351PC352G

R334C


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180°

Relative crosslinked product

0 0.5

G355

G357

G329

L266

S218

T222

V224

V269

M327

S65

Relative crosslinked product0 0.5 1.0 1.5 2.0

T142K151K161T168Q171T175D184S193E195E207

K220

I236A240D262L266V269T270N273D274

A281

N295

E309R305

Q316

A320E321E322C323M327G329P333R334P335G340L342E344Q345Q347E353G355N356G357L358F362D367E370E374

S218

T222V224N227

D280H279

L283Q282

G284Y285

L297K299E300

E310N313R314

Q317Y318

w/o

A


Q282

H279

K220

E344

97

64

6

L342 Q345 Q347 E353 G355 G357 L358 F362- + - + - + - + - + - + - + - +UV

BPA:

GST-Parkin(S65E)

*His-Ub(S65D)

T142 K161 Q171 T175 D184 E207 E344 N356- + - + - + - + - + - + - + - +

T168 S193 E195 L266 E322 C323 G329 G340- + - + - + - + - + - + - + - +UV

BPA:- + - + - + - + - + - + - + - +

K220 I236 A240 V269 T270 N273 E374w/o

GST-Parkin(S65E)

*His-Ub(S65D)

97

64

6

B

C

D

E

ArabinoseBPA - - +

+- +- - +

+- +noneamber K220

64

51

39

GST-Parkin(S65E)

*

*****

BPA:- + - + - + - +T270 M327 E344 N356

GST-Parkin(S65E)

*

His-Ub(S65D)

His-Ub(S65D)97

64

6

UVBPA:

GST-Parkin(S65E)

*His-Ub(S65D)

97

64

6

D274 A281 N295 Q316 M327 P333 R334 P335- + - + - + - + - + - + - + - +

K151 D262 E309 A320 E321D367 E370- + - + - + - + - + - + - +

UVBPA:

GST-Parkin(S65E)

*His-Ub(S65D)

97

64

6

E310 N313 R314 Q317 Y318- + - + - + - + - +

H279 Q282 R305- + - + - +

S218 T222 V224 N227 D280 L283 G284 Y285- + - + - + - + - + - + - + - +

L297 K299 E300- + - + - +UV

BPA:

GST-Parkin(S65E)

*His-Ub(S65D)

97

64

6

N356


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Cro

sslin

ked

prod

uct (

%)

0

30

20

10

40

50

60

BPA:

G32

9

w/o

L266

S21

8

T22

2

V22

4

V26

9

M32

7

G35

5

G35

7

-+ PINK1

PINK1E

DUb

PINK1UV - + - +- +- + - +

- +

- +

-- -+ ++

++ - +- +

w/o BPA M327BPA G329BPAL266BPA V269BPA

- +- +++ ++ ++

- +- + - + - +

++- +- +- +- +

++ ++ ++

- + - +- +- +

++ ++

V224BPA

- + - +- +- +

++ ++

Parkin:

UbPINK1

UV

Parkin:

none

64

97

39

14

51

28

6

GST-ParkinGST-PINK1

His-Ub

GST-ParkinGST-PINK1

His-Ub

64

97

39

14

51

28

6

- +- + - + - +

++- +- +

T222BPA S218BPA

- +- +++ ++ ++

A

BPA:

G32

9

w/o

L266

V26

9M

327

G35

5G

357

S21

8T

222

V22

4

64

97

39

14

51

28

6

GST-Parkin

Truncated Parkin

97

64

51

39

28

14

98

6249

38

28

6

14

GST-TcPINK1

His-Ub

B

C 40

30

20

10

0Frac.# 7 8 9 10 11 12 13 14 15

The

am

ount

of u

biqu

itin

(%

of t

otal

)

-PINK1+PINK1

Frac.# 7 8 9 10 11 12 13 14 15 7 8 9 10 11 12 13 14 15

64

98

146

GST-Parkin

GST-PINK1

phospho-GST-Parkin

His-Ub

His-Ub

phospho-His-Ub

75 29 13.7 6.5 kDa 75 29 13.7 6.5 kDa

-PINK1 +PINK1

16

6

GST-Parkin

G355BPA G357BPA

- +- + - + - +

++- +- +- +- +

++ ++ ++

64

97

39

14

51

28

6

IB: Parkin

IB: Ub



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62

38

14

49

28

6

BPA:

K11

P37

R42

R72w/o I44

Q49T

7 L8

G10

E34 I3

6

N60

62

38

14

49

28

6

E18

D32

F45

Q62Q

2

E51

R54

H68

V70

K63

T66F

4K

6E

16

E24

N25

D39

His-Ub

A

B

C D

His-Ub

GST-TcPINK1 9864

50

36

6

16phospho-His-Ub

PINK1 - + - +- +- + - + - +- +- + - + - + - + - +- +BPA:

His-Ub

GST-TcPINK1

phospho-His-Ub

PINK1

BPA:

K11 P37 R42 R72w/o I44 Q49 T7 L8 G10 E34 I36 N60

Pho

spho

ryla

ted

ubiq

uitin

(%

)

0

20

40

60

80

100

BPA: K11

P37

R42

R72w/o I44

Q49T

7 L8G

10

E34 I3

6

N60

986450

36

6

16

- + - +- +- + - + - +- + - + - + - + - +- +Q2 F4 K6 E16w/o E18 E24 N25 D32 D39 F45 E51 R54

- + - + - + - + - +- +Q62 K63 T66 H68 V70

Q2

F4

K6

E16

E18

E24

N25

D32

D39

F45

E51

R54

Q62

K63

T66

H68

V70

F45

R42

R72

I44

Q49

V70

S65



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Cro

sslin

ked

prod

uct (

%)

30

20

10

0

40

50

60

70

80

BPA:

K11w/o T7 L8

G10

E34 I3

6

K6

P37

Q62

K63

T66

H68

N60Q2

F4

E16

E18

D39

E51

R54

E24

N25

D32

-+ PINK1

PINK1

Cro

sslin

ked

prod

uct (

%)

30

20

10

0

40

50

70

60

80

BPA: R54 R42 F45 V70I36 R72I44 Q49

Parkin(WT) - Ub(WT)Parkin(S65E) - Ub(WT)Parkin(WT) - Ub(S65D)Parkin(S65E) - Ub(S65D)

180°

Crosslinked product

0 40K11

T7L8

G10

I36

K6

K63

E16R42

R72

S65

B

D

A

GST-ParkinGST-PINK1

ParkinPINK1

His-Ub

UV

- +-- -+ ++

++ - +- +

w/o BPA G10BPA K11BPA P37BPAT7BPA L8BPA E34BPA I36BPA

- +- +++ ++ ++ ++

- +- +- +- +++ ++ ++

- +- +++ ++ ++

- +- +- +- +++ ++ ++

- +- +++ ++

His-Ub: none

- + - +- +- + - + - + - +- + - + - + - + - + - +- + - + - + - + - +

64

97

39

14

51

28

6

64

97

39

14

51

28

6

F4BPA K6BPA E24BPAQ2BPA E16BPA E18BPA

- +- + - + - +

++- +- +- +- +

++ ++ ++

- + - +- +- +

++ ++

- +- + - + - +

++- +- +- +- +

++ ++ ++

- + - +- +- +

++ ++D39BPAN25BPA D32BPA

- +- + - + - +

++- +- +- +- +

++ ++ ++

- + - +- +- +

++ ++

GST-ParkinGST-PINK1

ParkinPINK1

His-Ub

UV

His-Ub:

GST-ParkinGST-PINK1

ParkinPINK1

His-Ub

UV

His-Ub: Q62BPAE51BPA R54BPA

- +- + - + - +

++- +- +- +- +

++ ++ ++

- + - +- +- +

++ ++H68BPAK63BPA T66BPA

- +- + - + - +

++- +- +- +- +

++ ++ ++

- + - +- +- +

++ ++

64

97

39

14

51

28

6

GST-Parkin

His-Ub

R42BPA I44BPA Q49BPA R72BPA

Parkin:Ub: WT

WT WTWT

S65E S65ES65D

S65E S65E

- + - + - + - + - +- + - + - + - + - + - + - + - +- + - + - +UV

S65D WTWT WT

WT S65DS65D WTWT WT

WTS65E S65E

S65DS65E S65E

S65D WTWT WT

WT S65D S65D

64

97

39

14

51

28

6

GST-Parkin

His-Ub

I36BPA R54BPA F45BPA V70BPA

Parkin:Ub: WT

WT WTWT

S65E S65ES65D

S65E S65E

- + - + - + - + - +- + - + - + - + - + - + - + - +- + - + - +UVS65D WT

WT WTWT S65DS65D WT

WT WTWT

S65E S65ES65D

S65E S65ES65D WT

WT WTWT S65D S65D

C

N60BPA

- + - +- +- +

++ ++

64

97

39

14

51

28

6

64

97

39

14

51

28

6

E



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B

C

97

64

97

64

51

28

39

14

6

Time 0 10 30 100 0 10 30 100 (min)

PINK1 PINK1- +

GST-Parkin

Ub-VS

0

5

10

15

20

25

0 20 40 60 80 100

PINK1-

PINK1+

Time (min)

Ub-

VS

con

juga

tion

effic

ienc

y (%

)

IB: Parkin

IB: HA (Ub-VS)

D

A 7 FNSSYGFPVEVDSDTSIFQLKEVVAK 27 (Parkin)

Sample # 1 2 3 4 5 6 7 8 9 10

4.40e64.00e63.60e63.20e62.80e62.40e62.00e61.60e61.20e68.00e5

0 04.00e5

9.86e68.77e67.67e66.58e65.48e64.38e63.29e62.19e61.10e6

1.32e71.21e71.10e7

Are

a350 VTCEGGNGLGCGFVFCR 366 (Parkin)

Sample # 1 2 3 4 5 6 7 8 9 100

1.48e71.30e71.11e79.28e67.42e65.57e63.71e61.86e6

2.04e71.86e71.67e7

0

5.58e74.89e74.19e73.49e72.79e72.09e71.40e76.98e6

7.68e76.98e76.28e7

Are

a

198 YIAWPLQGWQATFGGGDHPPK 218 (GST)

Sample # 1 2 3 4 5 6 7 8 9 10

3.04e82.76e82.48e82.21e81.93e81.66e81.38e81.10e88.28e75.52e72.76e7

0

1.02e99.33e88.48e87.63e86.79e85.94e85.09e84.24e83.39e82.54e81.70e88.48e7

0

Are

a

315 YQQYGAEECVLQMGGVLCPR 334 (Parkin)

Sample # 1 2 3 4 5 6 7 8 9 10

8.99e88.24e87.49e86.74e85.99e85.24e84.50e83.75e83.00e82.25e81.50e87.49e7

0

3.55e83.23e82.91e82.58e82.26e81.94e81.61e81.29e89.67e76.46e73.23e7

0

Are

a12 TITLEVEPSDTIENVK 27 (Ubiquitin)

Sample # 1 2 3 4 5 6 7 8 9 10

2.70e92.45e92.21e91.96e91.72e91.47e91.23e99.81e87.36e84.90e82.45e8

0

2.70e92.46e92.21e91.96e91.72e91.47e91.23e99.82e87.37e84.91e82.46e8

0

Are

a

GST-Parkin

GST-PINK1

PINK1UV

His-Ub:

64

97

64

97

I36BPA

- +-+-

+- +

GST-Parkin(S65E)

UV +- - + + +

none

His-Ub(S65D): noneR42BPA

R72BPA

3 41 2 7 8 9 1065

Ubl

RING0

RING1

IBR

REP RING2

Phosphorylated S65R42

R72

I36

Parkin (350-366 aa): VTCEGGNGLGCGFVFCR

Parkin (315-334 aa): YQQYGAEECVLQMGGVLCPR

ubiquitin

E18R170

K151

R305Phosphorylated

S65

S65

S65C431 C431



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0 20 40 60 80 100% of cells having hAG-Ub foci

Ash-ParkinhAG-Ub

K151A

A320R

V324R

A320R/V324R

R305A

K151A/R305A

R170E

K220E

R170E/K220E

WT

WTI44A

WT

WT

K220E R170E/K220E

K151A R305A R170E

A320R

Ub I44A

A320R/V324RV324R

K151A/R305AA

B

CD

WT K151A/R305A R170E/K220E A320R V324R

0 20 40 60 80% of cells having Parkin on mitochondria

YFP-Parkin

A320R

V324R

K151A/R305A

R170E/K220E

WT

YF

P-P

arki

nT

OM

M20

Mer

ged


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Hirokawa, Keiji Tanaka and Noriyuki MatsudaKoji Yamano, Bruno B. Queliconi, Fumika Koyano, Yasushi Saeki, Takatsugu

ActivationSite-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin

published online August 10, 2015J. Biol. Chem.

10.1074/jbc.M115.671446Access the most updated version of this article at doi:

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1 site-specific interaction mapping of phosphorylated ubiquitin to

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