Molecular Biology
The human PARK2 gene optimized for bacterial expression of FL-parkin was cloned in the pET15b vector. Various parkin mutations used in the present study were made using site-directed mutagenesis (SDM). TEV cleavage site (ENLYFQS) was substituted in the parkin construct between the 382nd -388th as described in 24, and an HRV 3C site (LEVLFQGP) was inserted between residue 140th and 141st residues using site-directed mutagenesis. Ubl (expressing 1-76th amino acids of parkin) and Ubl-linker (expressing 1-140th amino acids of parkin) constructs were generated by introducing a stop codon after the 76th, and 140th amino acids, respectively, in the FL-parkin construct. Ubl-linker-ΔACT (1-140, Δ101-109) construct was made on Ubl-linker using SDM. Miro1 (expressing 181st - 592nd amino acid) was amplified from the cDNA of the HEK293T cell line using Phusion polymerase (NEB) and cloned into the pGEX-6P1 vector using EcoRI and BamHI restriction enzymes. To generate fluorescently labeled ubiquitin, ubiquitin (residues 2-76) was cloned in a pGEX-6P vector with an overhang expressing GPLCGS at the n-terminal of ubiquitin. For the generation of ubiquitin-3Br protein, the ubiquitin gene (residues 1-75) was cloned in the pTXB-1 vector. Pediculus humanus corporis PINK1 (115 - 575) was a gift from David Komander 41(Addgene plasmid # 110750). Ube1 was a gift from Cynthia Wolberger 42 (Addgene plasmid # 34965).
Protein purification
Parkin constructs were expressed in Escherichia coli BL21(DE3)pLysS cells. Cells were grown until OD600 reached 0.4; the temperature was reduced to 16 ⁰C, and protein was induced by adding 50 µM IPTG, and media was supplemented with 200 µM ZnCl2. Cells were left to grow overnight at 16 ⁰C. Cells were harvested and lysed using sonication in lysis buffer (25 mM Tris pH 7.5, 200 mM NaCl, 5 mM Imidazole, 1 mM β-mercaptoethanol, and 100 µM AEBSF). Protein was purified over Ni-NTA resin. His-Sumo tag was removed using SENP1 protease. Protein was further purified over Hi-Trap Q HP column (GE Healthcare) followed by a gel-filtration column pre-equilibrated with storage buffer (25 mM Tris pH 7.5, 75 mM NaCl, 250 µM TCEP). Other proteins were also purified using similar protocols. PhPINK1 was purified as published before 41.
Isothermal Titration calorimetry
Isothermal titration calorimetry experiments were performed using Nano ITC (TA instruments). All titrations were performed at 25°C in a buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl and 250 µM TCEP. In Fig 2A, experiments were done using 350 µM of P-parkin (K211N) in the syringe and 25 µM of ΔUbl-parkin in the cell. In Fig. 2B, experiments were done using 220 µM of ΔUbl-parkin (RING2 untethered with TEV) in the syringe and 40 µM of P-parkin (K211N) in the cell. Results were analyzed in NanoAnalyze software using an independent mode of a binding model.
Ubiquitination assays
Ubiquitination assays were performed using fluorescently labeled ubiquitin. Ubiquitin labeling was done using DylightTM 800 Maleimide (Thermo Scientific), as mentioned in 26, using the manufacturer’s specifications. Ubiquitination reactions were performed at 25 ⁰C for 40 minutes in 25 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgCl2, and 0.1 mM DTT, 10 mM ATP. In all reactions, 25 nM Ube1, 250 nM UbcH7 (E2), 1 µM of E3, and 2 µM of UbIR800 were used in 20 µl of the total reaction volume. 0.5 µM of Ub or pUb was used as an allosteric activator for the experiments in Fig. 1F/4E, Extended Data Fig. 3B/3E. Increasing concentrations of pUbl (4 µM, 8 µM, 16 µM, 32 µM, and 64 µM) were used as trans activators in Fig. 2F. The transactivation experiments using pUbl, pUbl-linker, and pUbl-linker-ΔACT were carried out in increasing concentrations of 4 µM, 8 µM, and 16 µM in Fig. 3F, G, H. Miro1 substrate ubiquitination reaction was done at 25 ⁰C for 20 minutes with 5µM Miro1 and 0.5 µM of E3. Other conditions were the same as mentioned above for ubiquitination/autoubiquitination assay. The reactions were quenched by SDS loading dye and heated at 95⁰C for 5 mins. The samples were resolved on gradient SDS-PAGE and analyzed using Li-COR® Odyssey Infrared Imaging System. Each assay was repeated at least three times. ImageJ software was used for quantification of ubiquitination. Bar plots and statistical analysis were done using R.
Purification of phospho-Ubiquitin (pUb)-3Br
pUb-3Br was purified as published before 28,43. Briefly, Ubiquitin (1-75)-Mxe-intein-chitin binding domain was expressed using Escherichia coli BL21(DE3) cells using a pTXB-1 vector. Cells were induced at 0.8 O.D. with 250 µM IPTG and incubated at 22 ⁰C for 12 hrs. Cells were lysed in lysis buffer (20 mM Na2HPO4 pH 7.2, 200 mM NaCl, 0.1 mM EDTA) and purified using Chitin resin (NEB). The resin was incubated with cleavage buffer (20 mM Na2HPO4 pH 6.0, 200 mM NaCl, 50 mM MESNa, 0.1 mM EDTA) overnight to elute the protein. The eluted protein was reacted with 3-Bromopropylamine (Sigma) at 25 ⁰C for 4 hrs. The reacted protein was purified over Hiload 16/600 Superdex 75pg column (GE Healthcare) pre-equilibrated with 1X PBS. The fractions containing Ub-3Br were concentrated and phosphorylated using PhPINK1. pUb-3Br was purified over Hiload 16/600 Superdex 75pg column pre-equilibrated with parkin storage buffer.
Synthesis and purification of UbcH7~Ub
The reaction containing 500 µM of UbcH7 (Cys17Ser/Cys86Ser/Cys137Ser), 15 µM of Ube1, and 2.5 mM of 6xHis-Ub in charging buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 10 mM ATP) was incubated at 37 ⁰C for 18 hrs. The progress of the reaction was monitored over SDS-PAGE. The reacted sample was passed through Ni-NTA resin to capture His-Ub and UbcH7~Ub (His), and the eluted fraction was purified over Hiload 16/600 Superdex 75 pg column (GE Healthcare). Fractions containing UbcH7~Ub were pooled together and stored for further use.
Preparation of parkin complexes for crystallization
In the present study, parkin complexes with pUb were captured using pUb-3Br. To capture parkin complexes with pUb-3Br, human parkin constructs were mutated to include Gln347Cys, as published before 28, in various constructs for crystallization experiments. For crystallization of pUbl-linker (1-140) depleted parkin (141-465) and pUbl-linker (1-140) depleted parkin (141-465, Arg163Asp, Lys211Asn) recombinant constructs containing FL-parkin (HRV 3C site between 140th – 141st residue, TEV site between 382nd – 383rd, Gln347Cys) and FL-parkin (HRV 3C site between 140th – 141st residue, Arg163Asp, Lys211Asn, Gln347Cys) respectively, were made. Proteins were expressed and purified as above. Purified proteins were mixed with pUb-3Br, and parkin was phosphorylated using PhPINK1 in a phosphorylation buffer containing 5 mM ATP along with pUb-3Br. GST-HRV 3C protease was added (1:50), and proteins were left overnight at 4 ⁰C. The proteins were passed through affinity chromatography to remove GST-HRV 3C protease and PhPINK1. Flow-through was further purified over a gel-filtration column. Fractions containing R0RBR with pUb were pooled together and used for crystallization.
Ternary trans-complex of phospho-parkin (1-140 + 141-382 + pUb) was made using parkin (Lys211Asn, HRV 3C site between 140th – 141st residue) construct as the donor of pUbl-linker, and R0RBR (141-465, TEV site between 382nd – 383rd, Gln347Cys) construct as the acceptor of pUbl-linker. Purified parkin (Lys211Asn, HRV 3C site between 140th – 141st residue) was phosphorylated using PhPINK1 as above. Purified R0RBR (141-465, TEV site between 382nd – 383rd, Gln347Cys) was treated with His-TEV, and His-TEV was removed over Ni-NTA resin. 2-fold molar excess of phospho-parkin (Lys211Asn, HRV 3C site between 140th – 141st residue) was mixed with R0RBR (Gln347Cys, untethered RING2). The complex containing phospho-parkin (Lys211Asn, HRV 3C site between 140th – 141st residue) and R0RB (141-382, Gln347Cys) was purified over Hiload 16/600 Superdex 200pg column pre-equilibrated with parkin storage buffer. The latter complex was mixed with pUb-3Br and treated with 3C protease. Protein was further purified over Hiload 16/600 Superdex 75pg column pre-equilibrated with parkin storage buffer. Fractions containing ternary trans-complex of phospho-parkin (1-140 + 141-382 + pUb) were pooled together, concentrated, and used for crystallization.
Ternary trans-complex of phospho-parkin with cis ACT (1-76 + 77-382 + pUb) was made using the Ubl domain of parkin (1-76) and ΔUbl-parkin (77-465, TEV site between 382nd – 383rd, Gln347Cys). ΔUbl-parkin (77-465, TEV site between 382nd – 383rd, Gln347Cys) was treated with His-TEV, and His-TEV was removed over Ni-resin. A 3-fold molar excess of the pUbl domain was mixed with ΔUbl-parkin (Gln347Cys, untethered RING2). The complex containing pUbl and ΔUbl-parkin (77-382, Gln347Cys) was purified over Superdex 75increase 10/300 GL column pre-equilibrated with parkin storage buffer. The latter trans-complex of parkin with cis ACT (1-76 + 77-382) was mixed with pUb-3Br and purified again over Superdex 75increase 10/300 GL column pre-equilibrated with parkin storage buffer. Fractions containing ternary trans-complex of phospho-parkin with cis ACT (1-76 + 77-382 + pUb) were pooled together, concentrated, and used for crystallization.
Crystallization and structure determination
Initial crystals of pUbl-linker (1-140) depleted parkin (141-465) complex with pUb-3Br appeared in 1.6 M Ammonium sulfate, 0.1 M MES monohydrate pH 6.5, and 10% v/v 1,4-Dioxane of HR112 screen (Hampton Research) at 4°C. Seeding was done to grow bigger crystals in the same condition. The mother liquor containing 20% (v/v) of glycerol was used as a cryoprotectant for freezing crystals in liquid nitrogen. pUbl-linker (1-140) depleted parkin (141-465, Arg163Asp, Lys211Asn) complex with pUb-3Br crystals appeared in 0.15 M Potassium bromide, and 30% w/v Polyethylene glycol monomethyl ether 2,000 of Index screen (Hampton research) at 18°C. The mother liquor containing 20% (v/v) of PEG 400 was used as a cryoprotectant for freezing crystals in liquid nitrogen. Crystals of ternary trans-complexes of phospho-parkin were obtained in 0.3 M Sodium nitrate, 0.3 Sodium phosphate dibasic, 0.3 M Ammonium sulfate, 0.1 M Tris (base) & BICINE (pH 8.5), 25% v/v MPD, 25% w/v PEG 1000, and 25% w/v PEG 3350 of Morpheus screen (Molecular dimensions). Good quality crystals were grown at 18 °C using microseeding. The mother liquor containing 10% (v/v) of glycerol was used as a cryoprotectant for freezing crystals in liquid nitrogen.
Data were collected at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Data were processed using XDS 44. Scaling was done using Aimless, and the structure was determined by molecular replacement using Phaser as implemented in CCP-7.1 45. Structures of pUbl-linker (1-140) depleted parkin (141-465) or pUbl-linker (1-140) depleted parkin (141-465, Arg163Asp, Lys211Asn) in complex with pUb-3Br were solved by using Pediculus parkin structure in complex with phospho-ubiquitin (PDB 5CAW) as a search model. Structures of ternary trans-complex of phospho-parkin were solved using phospho-parkin structure (PDB 6GLC) as a search model. The initial model was built and refined using coot 46 and refmac5 47.
Purification of phosphorylated proteins
PhPINK1 was used to phosphorylate various parkin variants used in the study. Phosphorylation buffer contains 50 mM Tris pH 8.5, 100 mM NaCl, 10 mM MgCl2, 10 mM DTT, and 10 mM ATP. The reactions were performed at 25 °C for 4 hrs. Phosphorylation status was checked using Phos-Tag (FUJIFILM) analysis as per the manufacturer’s protocol. PINK1 was depleted by affinity chromatography upon completion of the reaction. The phosphorylated proteins were further purified over a gel-filtration column.
Parkin phosphorylation assay
Parkin phosphorylation assay was performed using 5 µM parkin and 0.25 µM PINK1 in phosphorylation buffer at 25 °C for 15 mins. Increasing concentrations (20 µM, 40 µM, and 80 µM) of pUbl or pUb were added with parkin to check their effect on parkin phosphorylation. The samples were analyzed using Phos-Tag (FUJIFILM) analysis using the manufacturer’s protocol.
Size-exclusion chromatography analysis
For RING2 or Ubl displacement/binding assays, HRV-3C cleavable and TEV cleavable constructs of parkin were purified and phosphorylated as above. TEV and HRV 3C were added at the molar ratio (protease: parkin) of 1:5 and 1:15, respectively. After incubation with respective proteases, proteins were purified using affinity chromatography to remove proteases from parkin. The proteins were loaded onto Superdex 75increase 10/300 GL column, and fractions were analyzed using SDS-PAGE.
For the trans-complex assays, phospho-parkin variants were added in 2-fold molar access. Also, in all trans-complex assays, the TEV site between IBR and RING2 was present only on the target molecules. Furthermore, before complex formation, TEV was removed by affinity chromatography. Proteins were incubated for 30 minutes at 4 ⁰C before loading onto Superdex 75increase 10/300 GL column. Fractions were analyzed using SDS-PAGE.
For SEC assay to analyze parkin interaction with E2~Ub, 10 µM of phospho-parkin/phospho-parkin-ΔACT/phospho-parkin Ile411Ala was pre-incubated with 15 µM of pUb, followed by the addition of 20 µM of E2~Ub. Proteins were incubated for 1 hr at 4⁰C before injecting onto Superdex 75increase 10/300 GL column. Fractions were analyzed using SDS-PAGE to check the complex formation.