Nature Structural & Molecular Biology doi: /nsmb Supplementary Figure 1. CRBN binding assay with thalidomide enantiomers.

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1 Supplementary Figure 1 CRBN binding assay with thalidomide enantiomers. (a) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were washed three times with 0.5% NP-40 lysis buffer and bound proteins were eluted with wash buffer containing 1 mm S-, R-thalidomide (S-Thal or R-Thal) or 0.1% DMSO for the indicated time. The eluate was then analysed by SDS-PAGE and immunoblotting (IB). (b) As in a but eluted with a buffer solution containing the indicated concentrations of S or R-thalidomide (S- or R-Thal). (c) Inhibitory effects of thalidomide

2 enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of S- or R-thalidomide for 4 hours prior to harvesting. Supplementary Figure 2 The thalidomide-binding domain (TBD) of mouse Cereblon showing the crystal contacts formed between protein monomers, bridged by thalidomide molecules. Chain B is shown in orange and chain D is shown in yellow.

3 Supplementary Figure 3 Structural comparison between the TBD tri-trp pockets. (a) Comparison between thalidomide bound and apo CRBN showing that the binding pocket is formed in the absence of IMiD. The thalidomide bound form is shown with carbons in cyan/yellow and the apo form is shown with carbons in grey. Comparison of the Tri- Trp hole with related modified amine-binding sites. (b), The trimethyl lysine-binding pocket of HP1 chromodomain (grey). The pocket is composed of one Trp and two Tyr residues for binding to histone H3 trimethylated Lys. HP1 is a member of the royal family group of proteins which possess an aromatic methylated lysine and/or arginine-binding pocket. The binding pocket is usually composed of two to four aromatic residues, providing electrostatic and hydrophobic contacts to accommodate the insertion of methylated ligand of the binding partner proteins. The pocket of BPTF PHD finger is composed of one Trp and three Tyr residues for binding to histone H3 trimethylated Lys42(not shown). (c) The dimethyl lysine-binding pocket of 53BP1 Tudor domain (grey) of the royal family. The pocket is formed by one Trp, two Tyr, one Phe and one Asp residue (green), forming an aromatic environment with a salt bridge (dotted line) between the Lys dimethyl amino group and the Asp side-chain carboxyl group. (d) The acetyl lysine-binding pocket of GCN5 bromodomain (grey). The pocket is formed by a mixture of aromatic (three Tyr and one Phe), aliphatic (Val and Pro) and Asn residues (green). The acetyl group is recognized by formation of a direct hydrogen bond to Asn and water-mediated hydrogen bonds (broken lines). (e) Overlay of the tryptophan box forming the betaine-binding pocket of E. coli ProX (green) on the Tri-Trp hole ofpocket of the CRBN TBDMBS domain (cyan). Three Trp residues are labeled. S-thalidomide (SThal, yellow) bound to the Tri-Trp hole pocket is also shown. (f) The betaine-binding site of E. coli ProX (grey). The tryptophan box formed by three conserved Trp and one Tyr residue (green) creates an aromatic environment for binding to betaine (N,N,Ntrimethyl glycine) by cation-pi interactions and van der Waals contacts. In contrast to the CRBN Tri-Trp pocket, the ProX betaine-binding site is located at the cleft between two domains, and the Trp residues of the tryptophan box and the bound betaine are completely occluded inside the protein. (g) As in f, but for the betaine-binding pocket of E. coli BetP (grey). The Trp residues and the bound betaine are completely occluded inside the protein as in ProX.

4 Supplementary Figure 4 Structural superposition of Cereblon TBD with homologs. Cereblon TBD is shown in blue, methionine sulfoxide reductase is shown in green and RIG-I in magenta.

5 Supplementary Figure 5 Immunoblot and immunohistochemical quantiification of CRBN. (a) Immunoblot analysis of CRBN protein in lysates from DF15, DF15R, DF15R RFP (RFP Ctrl), DF15R CRBNWT (CRBN wt), DF15R CRBN W386A and CRBN W400A cells. (b) CRBN analysis in DF15 and DF15R and DF15R derived cell lines by immunohistochemistry. Images were obtained using a Olympus BX45 microscope at a 40x objective. CRBN signal is shown as brown color and hematoxylin counterstain identifies the nucleus of cells. (c) Immunoblot of anti-flag immunoprecipitation from cell extracts expressing Flag-tagged CRBN proteins. (d) Immunoblot of thalidomide analog affinity bead binding to CRBN in DF15, DF15R and DF15R CRBNWT cell extracts. Lane description in order left to right: In = DF15 input prior to bead purification; V = DF15 extract control (1% DMSO preincubation); L = DF15 extract preincubated with lenalidomide (30 µm); P = DF15 extract preincubated with Pomalidomide (30 µm); In = DF15R input prior to bead purification; V = DF15R control (1% DMSO preincubation); L = DF15R extract preincubated with lenalidomide (30 µm). P = DF15R extract preincubated with Pomalidomide (30 µm); In = DF15R CRBNWT input prior to bead purification; V = DF15R CRBNWT control (1% DMSO preincubation); L = DF15R CRBNWT extract preincubated with lenalidomide (30 µm). P = DF15R CRBNWT extract preincubated with Pomalidomide (30 µm); Representative immunoblot from two independent experiments with similar results.

6 Supplementary Figure 6 IL-2 co-stimulation by pomalidomide in human PBMCs but not in mouse splenocytes. (a) Co-stimulation of IL-2 release by pomalidomide in human PBMC cells treated with anti-cd3. Data shown as means ±s.d.. (b) Costimulation of IL-2 release by anti-cd28 (red) or pomalidomide (blue) in mouse PBMC cellssplenocytes treated with anti-cd3. Data shown as means ±s.d.