Reagents and antibodies
(S/R)–thalidomide (Sigma-Aldrich and Tokyo Chemical Industry Co., Ltd.), (S)-thalidomide (Sigma-Aldrich), (R)-thalidomide (Sigma-Aldrich), pomalidomide (Sigma-Aldrich), lenalidomide (FUJIFILM Wako Pure Chemical Corporation), (S/R)-5HT were prepared according to a previously published method45. (S)-5HT, (R)-5HT, and (S/R)-5HT (Enamine) at 50 mM each were dissolved in dimethyl sulfoxide (DMSO; FUJIFILM Wako Pure Chemical Corporation) and stored at ‒20 °C as stock solutions. All the drugs were diluted 1000-fold for the cell degradation analyses or diluted 200-fold for the AS-based analyses using the AS technology. For the crystallization and ITC experiments, (S)-thalidomide and (S)-5-hydroxythalidomide at 1 M were dissolved in DMSO as stock solutions.
In this study, the following antibodies were used at each dilution ratio: anti-FLAG mouse monoclonal antibody (mAb) (1:5000, horseradish peroxidase (HRP)-conjugated, Sigma-Aldrich, A8592), anti-AGIA rabbit mAb46 (1:10,000, HRP-conjugated, produced in our laboratory), and anti-Myc mouse mAb (1:3000, HRP-conjugated, Cell Signaling Technology, 2276) were used for the detection of epitope-tagged proteins. Anti-α-tubulin rabbit polyclonal Ab (1:10,000, HRP-conjugated, MBL, PM054-7) was used to detect α-tubulin. Biotinylated proteins were detected by anti-biotin (1:5000, HRP-conjugated, Cell Signaling Technology, 7075).
Plasmids for the AS-based and cell degradation assays
pDONR221 and pcDNA3.1(+) plasmids, based on Gateway technology, were purchased from Invitrogen, and the pEU vector for the wheat cell-free system was constructed in our laboratory47. pcDNA3.1(+)-FLAG-GW, pcDNA3.1(+)-AGIA-MCS, pcDNA3.1(+)-Myc-MCS, and pEU-bls-GW plasmids were constructed based on each original vector by using the In-Fusion system (TaKaRa Bio) or restriction enzymes. pEU-FLAG-GST-IKZF1 and pEU-FLAG-GST-SALL4 were purchased from the Kazusa DNA Research Institute. Open reading frames (ORFs) in SALL4 and IKZF1 were amplified and restriction enzyme sites were added by PCR and cloned into pcDNA3.1(+)-AGIA-MCS or pcDNA3.1(+)-Myc-MCS. The ORF of CRBN was purchased from the Mammalian Gene Collection48. CRBN was amplified, and the BP reaction sequence (attB and attP) was added by PCR and cloned into pDONR221 using BP recombination (Invitrogen). Then, pDONR221-CRBN was recombined into pEU-bls-GW or pcDNA3.1(+)-FLAG-GW using LR recombination (attL and attR). Amino acid mutation in each protein was generated by inverse PCR and In-Fusion.
Protein expression and purification
DNA sequences encoded human CRBN TBD (318‒426 and C366S mutation) and SALL4 ZF2 (410‒432) were cloned into pGEX6P-3 (GE Healthcare), and the recombinant proteins were expressed in Escherichia coli Rossetta(DE3) (Novagen) using lysogeny-broth media supplemented with 20 μM ZnCl2, 100 μg ml‒1 ampicillin, and 17 μg ml‒1 chloramphenicol. Protein expression was induced by adding 0.5 mM isopropyl β-d-thiogalactopyranoside at 18 °C when the optical density at 600 nm (OD600) reached ~0.6. The cells were collected by centrifugation and were then resuspended in buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 0.1 mM tris(2-carboxyethyl)phosphine (TCEP). After sonication and centrifugation, the supernatant of the cell lysate was passed over glutathione Sepharose 4B resin (GE Healthcare), and resin-bound protein was cleaved overnight by human rhinovirus 3C protease. Proteins eluted from the resin were purified by size-exclusion chromatography with Superdex 75 10/300 GL (GE Healthcare) in 50 mM HEPES-NaOH, pH 7.4, 200 mM NaCl, and 0.1 mM TCEP. The fractions containing the CRBN TBD or SALL4 ZF2 were pooled and concentrated by ultrafiltration with Vivaspin 20 (MWCO 3000, Sartorius) to ~750 μM for the CRBN TBD and 100‒150 μM for SALL4 ZF2. The proteins were stored at ‒80 °C. The concentration of the CRBN TBD was determined by measuring the absorbance at 280 nm, and the molecular extinction coefficient was 27,960 M−1 cm−1 using Bradford protein assay kit (Thermo Fisher Scientific) was used to measure the concentration of SALL4 ZF2, with bovine serum albumin (BSA) serving as the protein standard.
For the AS-based interaction assay, recombinant SALL4–CRBN and IKZF1 were synthesized using a wheat cell-free system. In vitro transcription and translation based on wheat cell-free protein synthesis were performed using a WEPRO1240 expression kit (Cell-Free Sciences). Transcription reactions were conducted by SP6 RNA polymerase using DNA fragments as templates. The translation reactions were performed by the bilayer method using a WEPRO1240 expression kit according to the manufacturer’s protocol. For the synthesis of biotinylated CRBN, 1 µl of cell-free synthesized crude biotin ligase (BirA) was added to the bottom layer, and 0.5 µM (final concentration) of d-biotin (Nacalai Tesque) was added to both the top and bottom layers49.
Crystallization and data collection
The CRBN TBD and SALL4 ZF2 complex was crystallized by sitting-drop vapor diffusion in the presence of (S)-thalidomide or (S)-5HT. The SALL4–CRBN complex solution (104 μM) was prepared by mixing SALL4 ZF2 and the CRBN TBD with 2 mM (S)-thalidomide. The solution was mixed with an equal volume of a reservoir solution containing 27% (w/v) polyethylene glycol (PEG) 4000, 0.1 M sodium acetate, pH 5.5, and 0.1 M MgCl2, which was then equilibrated against the reservoir solution at 20 °C. For the (S)-5HT-bound SALL4–CRBN complex (70 μM), SALL4 ZF2 was mixed with the CRBN TBD and 1.8 mM (S)-5HT. The complex solution was further mixed with an equal volume of a reservoir solution containing 22% (w/v) PEG 4000, 0.1 M MES-NaOH, pH 6.0, and 0.2 M Li2SO4, which was then equilibrated against the reservoir solution at 20 °C.
Each obtained crystal was soaked in the reservoir solution containing 25% (v/v) ethylene glycol as a cryoprotectant using dual thickness MicroMounts (MiTeGen) and then cooled in a liquid nitrogen stream. Diffraction data were collected using a Pilatus 2M-F with an AR-NE3A beamline at the Photo Factory (Tsukuba, Japan). The data were indexed and integrated using XDS50 and scaled using AIMLESS51. The crystals of the SALL4–CRBN complex belonged to the space group C2221 with unit cell parameters of a = 70.96, b = 92.72, and c = 43.99 Å for (S)-thalidomide, and a = 83.62, b = 93.89 and c = 43.68 Å for (S)-5HT. The data collection statistics are summarized in Supplementary Table 1.
Structure determination and refinement
The ternary structures of SALL4 ZF2, CRBN TBD, and (S)-thalidomide were determined by molecular replacement with Phaser-MR of the PHENIX program suite52 using human CRBN TBD (Protein Data Bank (PDB) code: 4TZ4) and IKZF1 (PDB code: 6H0F) as search models. The ternary structures of the CRBN TBD, SALL4 ZF2, and (S)-5HT were determined by molecular replacement in the SALL4–CRBN complex with (S)-thalidomide, as determined in the present study. The iterative model building and refinement cycles were performed using COOT53 and phenix.refine54. All structures were generated with PyMOL (Schrödinger), and the model quality was evaluated with MolProbity55. The refinement statistics are summarized in Supplementary Table 1.
The binding affinity of (S)-5HT to the CRBN TBD was measured by using an isothermal titration calorimeter (MicroCal iTC200, Malvern) with a reference power of 5 μcal s‒1 and stirring speed of 750 r.p.m. at 25 °C. The CRBN TBD (wild-type or H353A mutant) was dialyzed in a binding buffer containing 50 mM sodium phosphate, pH 7.4, 200 mM NaCl, and 0.1 mM TCEP, and then DMSO was added to the protein solution at a final concentration of 0.2%. (S)-5HT or (S)-thalidomide was dissolved in DMSO, and the solution was mixed with binding buffer with the DMSO concentration adjusted to 0.2%. For titrations, the (S)-5HT solution (200 μM) or (S)-thalidomide (400 μM) was injected into the sample cell filled with the CRBN TBD solution (10 or 20 μM) in 37 consecutive 1.0-μl aliquots at 120-s intervals. The first injection volume was 0.4 μl, and the observed thermal peak was excluded from the data analyses. Data fitting was performed using the Origin 7.0 software (OriginLab) in the one set of sites mode. The values of the dissociation constant (KD) and molar binding ratio (N) were calculated with mean values ± SD (n = 3 independent experiments).
AS-based interaction assays
For this assay, we directly used translational mixtures from the wheat cell-free protein production system. Then, 0.5 µl of biotinylated CRBN and 0.8 µl of FLAG-GST-SALL4 or FLAG-GST-IKZF1 was mixed in 15 µl of AS buffer containing 100 mM Tris (pH 8.0), 0.01% Tween-20, 100 mM NaCl, and 1 mg ml−1 BSA. Then, 5 µl of a mixture containing 0.0125 µl of IMiD in AS buffer was added, and 20 µl of the mixture was incubated at 26 °C for 1 h in a 384-well AlphaPlate (PerkinElmer). Next, 5 µl of detection mixture containing 0.2 µg ml−1 anti-DYKDDDDK mouse mAb (dilution ratio of 1:2500, FUJIFILM Wako Pure Chemical Corporation), 0.08 µl of streptavidin-coated donor beads, and 0.08 µl of protein A-coated acceptor beads (PerkinElmer) in AS buffer was added to each well. After incubation at 26 °C for 1 h, luminescence signals were measured using an Envision plate reader (PerkinElmer).
Cell culture and transfection
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (low glucose) (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FUJIFILM Wako Pure Chemical Corporation), 100 U ml−1 penicillin, and 100 µg ml−1 streptomycin (Gibco) at 37 °C and 5% CO2. HEK293T cells were transfected using PEI Max: polyethyleneimine “Max” (MW 40,000) (PolyScience, Inc.).
Generation of CRBN-knockout (KO) HEK293T cells
For the generation of CRBN-KO HEK293T cells, the guide nucleotide sequence 5′-ACTCCGGGCGGTTACCAGGC-3′ was selected from the human CRBN gene. The CRBN-KO HEK293T cells were generated as previously published method56 by CRISPR/Cas9-mediated genome editing.
IMiD-induced proteasomal degradation assay
For experiments using mutant CRBN, the HEK293T-CRBN−/− cells were cultured in 48-well plates and transfected with 200 ng of pcDNA3.1(+)-FLAG-CRBN-WT or 200 ng of pcDNA3.1(+)-FLAG-CRBN-H353A and 15 ng of pcDNA3.1(+)-AGIA-SALL4. After incubation for 6 h, the cells were treated with DMSO (0.1%), thalidomide, or 5HT in culture medium at the indicated concentration for 18 h.
For experiments using SALL4 mutants, HEK293T-CRBN‒/‒ cells were cultured in 48-well plates and transfected with 200 ng of pcDNA3.1(+)-FLAG-CRBN-WT and 15 ng of pcDNA3.1(+)-AGIA-SALL4-WT or 15 ng of pcDNA3.1(+)-AGIA-SALL4-mutant. After incubation for 6 h, the cells were treated with DMSO (0.1%), thalidomide, or 5HT in culture medium at the indicated concentration for 18 h.
For experiments using mutant IKZF1, the HEK293T-CRBN‒/‒ cells were cultured in 48-well plates and transfected with 200 ng of pcDNA3.1(+)-FLAG-CRBN-WT and 15 ng of pcDNA3.1(+)-AGIA-IKZF1-WT or 15 ng of cDNA3.1(+)-AGIA-IKZF1-mutant. After incubation for 6 h, the cells were treated with DMSO (0.1%), thalidomide, or 5HT in culture medium at the indicated concentration for 18 h.
For experiments using 5HP, the HEK293T-CRBN−/− cells were cultured in 48-well plates and transfected with 200 ng of pcDNA3.1(+)-FLAG-CRBN-WT, 15 ng of pcDNA3.1(+)-AGIA-SALL4-WT, and 15 ng of pcDNA3.1(+)-Myc-IKZF1-WT. After incubation for 6 h, the cells were treated with DMSO (0.1%), thalidomide, or 5HT in culture medium at the indicated concentration for 18 h.
For all experiments, the cells were lysed by boiling in 1× sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), and 10% glycerol) containing 5% 2-mercaptoethanol.
Protein lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). After the membranes were blocked using 5% skim milk (Megmilk Snow Brand) in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) at room temperature for 1 h, the appropriate antibody was used. Immobilon (Millipore) or ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) was used as the HRP substrate, and the luminescence signals were detected using an ImageQuant LAS 4000 mini (GE Healthcare). To perform reprobing, stripping solution (FUJIFILM Wako Pure Chemical Corporation) was used, and the membranes were reblocked using 5% skim milk in TBST. The immunoblot data were analyzed by using ImageJ (version 2.0.0-rc-43/1.50e).
Further information on research design is available in the Nature Research Reporting Summary linked to this article.