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Supplementary Information Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2019 Supplementary Information Activity-based probe developed by sequential dehydroalanine formation strategy targets HECT E3 ubiquitin ligases Ling Xu,a, # Jian Fan,a, # Yu Wang,a,b Zhongping Zhang,c Yao Fu,a Yi-Ming Li,b* Jing Shi,a* a Department of Chemistry, University of Science and Technology of China; Hefei 230026, China, E-mail: shijing@ustc.edu.cn b School of Food and Biological Engineering, Hefei University of Technology, Hefei, Anhui 230009, China, E-mail: ymli@hfut.edu.cn; c Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, China ‡ These authors contributed equally to this work. Table of Contents 1. General Information………………………………………………………………….…… S2 a. Materials b. HPLC and FPLC c. Molecular biochemistry d. Mass spectrometry e. Protein Expression and Purification 2. Experimental Section……………………………………………………………….……. S4 a. Chemical synthesis of Thz-Cysteine-thiol 4 b. Protein expression and purification c. Synthesis of biotinylated Ub(1-75)-CONHNH2 d. Synthesis of E2-Ub-Dha probe 9 e. Synthesis of E2-Ub conjugate 10 f. Protein folding and purification. g. Circular dichroism spectra determination h. Cross-linking between E2-Ub-Dha and GST-HECT of UBE3C and NEDD4 i. Proteins captured from Hela cell lysates j. LC-MS/MS identification E3 ligases captured by E2-Ub-Dha probe 3. Experimental figures……………………………………………………………….……. S9 S1 General Information a. Materials NaCl, NaNO2, Na2HPO4·12H2O, imidazole, guanidine hydrochloride (Gn·HCl) were purchased from Sinopharm Chemical Reagent. Tris(2-chloroethyl) phosphate (TCEP), 4-mercaptophenylacetic acid (MPAA), N,N-Diisopropylethylamine (DIEA) were purchased from Aladdin (Shanghai, China). N,N-dimethylformamide (DMF), dichloromethane (DCM) were bought from Chengdu Kelong Chemicl Co., Led. Ttrifluoroacetic acid (TFA) (HPLC grade), 2-mercaptoethanesulfonic (Mesna) were bought from Energy Chemical. Triisopropylsilane (TIPS) were purchased from Ouhe Technology (Beijing, China). Acetonitrile (HPLC grade) was purchased from J. T. Baker (Phillipsburg, NJ, USA). b. HPLC and FPLC For protein analysis, analytical RP-HPLC (SHIMADZU, Prominence LC-20AT) with an analytical welch ultimate XB-C18 (250 mm×4.6 mm, 5 μm particle size, flow rate 1.0 mL/min, rt) or C4 (250 mm×4.6 mm, 5 μm particle size, flow rate 1.0 mL/min, rt) was used. For protein purification, semi-preparative XB-C18 (150×10 mm, 5 μm particle size, flow rate 4.0 mL/min, rt) or C4 (150×10 mm, 5 μm particle size, flow rate 4.0 mL/min, rt) was used. All analytical and semi-preparative injections were both monitored at UV absorption of 214 nm and 254 nm. The recombinant expressed protein and the refolded E2-Ub-Dha probe were separated by a GE Healthcare (AKTA Purifier 10 UPC-F920) instrument. The Superdex 75 column was used to separate the refolded probe, while the Superdex 200 column was used to separate the expressed proteins. c. Molecular biology and biochemistry UBE2D2* and GST-HECT of NEDD4 and UBE3C plasmids were designed and bought from Genscript Biotech Corp. Bacterial cells were grown in LB agar medium (Sigma) or LB (Luria-Bertani) medium. For SDS-PAGE, samples were loaded onto ExpressPlus™ PAGE gel (10×8, 10%, 12 wells) and electrophoresed for 35 min at 160 V. All pictures of protein gels and western were taken on Bio-Rad ChemDocTM Touch imaging system. d. Mass Spectrometry High-resolution ESI mass spectra and Agilent 6210 Time of Flight mass spectra were used to characterize the products. For LC-MS/MS analysis, Thermo LTQ Orbitrap Velos Pro was used to S2 identify the proteins captured by E2-Ub-Dha probe. e. Protein Expression and Purification UBE2D2* were incorporated in modified pET28a vectors and GST-HECT of UBE3C and NEDD4 were incorporated in pGEX-6P-1. All proteins were expressed in E. coli. BL21 (DE3) cells. Cells were then harvested using a centrifuge purchased from Sigma and lysed by sonication. S3 1. Experimental Section a. Chemical synthesis of Thz-Cysteine-thiol 4 The synthesis procedures were similar as Brik et al. reported1 Boc-4-L-thiazolidine-4-carboxylic acid (466.56 mg, 2 mmol) were dissolved in DMF (10 mL). Then, N-hydroxysuccinimide (NHS, 460.36 mg, 4 mmol), HBTU (670.58 mg, 2.4 mmol) and N,N-Diisopropylethylamine (DIEA, 1.26 mL, 8 mmol) were added. After stirring for 5 min, a solution of 2-(Tritylthio) ethanamine (766.73 mg, 2.4 mmol) in DMF (2 mL) was added dropwise. After vigorously stirring for 12 h at RT, the reaction mixture was diluted with EtOAc. The combined organic phase was washed with water and brine, dried over Na2SO4, filtrated and concentrated. The crude product was purified by chromatography to afford (1.26 mmol, 63%) as colorless oil. 1H NMR (400 MHz, Chloroform-d) δ 7.41 (t, 3H), 7.39 (t, 3H), 7. – 7.31 (m, 7H), 7.21 (m, 3H), 6.66 (s, 1H), 4.65 (d, J = 9.4 Hz, 2H), 4.32 (d, 1H), 3.36 (s, 1H), 3.17 (s, 1H), 3.07 (s, 2H), 2.39 (t, J = 6.4 Hz, 2H), 1.42 (s, 9H). The residue was dissolved in THF: DCM (1:1, 5 mL), followed by addition of triisopropylsilane (0.2 mL). The reaction mixture was stirred at room temperature for 1 h. After completion of the reaction (monitored by TLC), the organic phase was concentrated under vacuum. The remaining residue was dried under high vacuum overnight and used for the next step without further purification. b. Protein expression and purification UBE2D2*, UBE2D2 (C85K) (also refered as UBCH5B(C85K)), avitag-fused Ub and GST-HECT of UBE3C and NEDD4 were transformed separately into E. coli. BL21 (DE3) cells for protein expression. The cells were grown at 37℃ until the OD600 reached 0.8, then 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and the cells were induced at 16℃ for 18 hours. The cells were harvested by centrifugation at 4000 rpm for 25 min and further lysed by sonication with a lysis buffer (20 mM Tris, 500 mM NaCl, pH7.4) containing 1mM pheylmethylsulfonyl fluoride (PMSF). After centrifugation at 12000 rpm for 30 min, the supernatant solution was preserved. UBE2D2* and UBE2D2 (C85K) were further purified by Ni-NTA resin. Firstly, the resin was S4 washed three times with water and buffer (20 mM Tris, 500 mM NaCl) respectively. Then the supernatant solution was loaded onto the resin with a flow rate controlled at around 30 mL/h. The resin was washed with a buffer (20 mM Tris, 500 mM NaCl) containing 20 mM imidazole. Finally, the eluate was collected with a buffer (20 mM Tris, 500 mM NaCl) containing 250 mM imidazole for further HPLC purification. 1% perchloric acid was added into the supernatant solution of avitag-fused Ub, and most of the undesired protein was precipitated, while the biotinylation of ubiquitin was retained. Then the supernatant was dialyzed into the buffer (20 mM Tris, 500 mM NaCl). GST-HECT of UBE3C and NEDD4 were purified by GST agarose. The resin was washed with water and buffer (20 mM Tris, 500 mM NaCl) similar as above and the extract was loaded. Then the resin was washed with the buffer (20 mM Tris, 500 mM NaCl) and collected with the buffer (20 mM Tris, 500 mM NaCl) containing 20 mM reduced GSH. The proteins were concentrated to a final concentration 20 mg/mL and the buffer was exchanged with 50 mM Tris buffer (pH 7.6) containing 0.1 mM EDTA and 1 mM TCEP by size-exclusion chromatography (SEC) of gel filtration (Superdex 200). c. Synthesis of biotinylated Ub(1-75)-CONHNH2 Initially, N-terminal biotinylated Ub(1-45)-NHNH2 and Ub(46C-75)-NHNH2 segments were generated by solid phase peptide synthesis (SPPS). Specifically, the 2CTC resin was firstly converted into hydrazide resin with DMF solution containing 5% NH2NH2·H2O. Then the two segments of Ub(1-45)-NHNH2 and Ub(46C-75)-NHNH2 were synthesized by multiple rounds of Fmoc deprotection and amino acid coupling.2 The biotin tag was added to the N-terminus of Ub(1-45)-NHNH2 according to the amino acid addition step but dissolved in DMSO. Finally, the two segments were acquired by removing the side-chain protection using a cocktail (TFA / phenol / water / thioanisole / EDT, 85 : 2.5 : 5 : 5 : 2.5). The crude products were purified by HPLC. 5.4 mg N-terminal biotinylated Ub(1-45)-NHNH2 was dissolved in 900 µL PBS buffer （6 M Gn·HCl, 200 mM Na2HPO4）， pH 3. Then, 10 eq of NaNO2 was added to the buffer at -10℃. 30 min later, 30 eq of MPAA was added and pH was adjusted to 5.3. After 5 min, 3.4 mg Ub(46C-75)-CONHNH2 was added and the pH was adjusted to 6.5 at room temperature. The mix buffer was stirred about 3 h to afford N-terminal biotinylated Ub(1-46C-75)-NHNH2. After purified by HPLC, 18 mg N-terminal biotinylated Ub(1-46C-75)-NHNH2 was dissolved in 1 mL S5 PBS buffer (pH 7.0) containing 100 mM TCEP. 20 mM VA044 and 40 mM reduced GSH were further added to the buffer to conduct desulfurization at 37℃ for 8 h to give the final product 7 with 72% yield (13 mg). d. Synthesis of E2-Ub-Dha probe Firstly, 11.5 mg UBE2D2* 1 was dissolved in 0.65 μL 6M Gn·HCl, and 7.6 mg bisamide 2 dissolved in 50 μL DMF. This was added to UBE2D2* and the pH was adjusted to 8.0. The reaction was stirred for 2 h at 37 ºC, and the mixture was separated by HPLC using C18 semi-preparative column to obtain the product 3 with 83% yield (9.5 mg).
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