Structure of the human ​Cereblon–​DDB1–​lenalidomide complex reveals basis for responsiveness to thalidomide analogs

Journal name:
Nature Structural & Molecular Biology
Volume:
21,
Pages:
803–809
Year published:
DOI:
doi:10.1038/nsmb.2874
Received
Accepted
Published online

Abstract

The Cul4–​Rbx1–​DDB1–​Cereblon E3 ubiquitin ligase complex is the target of ​thalidomide, ​lenalidomide and ​pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind ​Cereblon (​CRBN) and promote the recruitment of substrates ​Ikaros (​IKZF1) and ​Aiolos (​IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human ​CRBN bound to ​DDB1 and the drug ​lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of ​CRBN accommodates the ​glutarimide moiety of ​lenalidomide, whereas the ​isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with ​thalidomide or ​pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.

At a glance

Figures

  1. The structures of thalidomide analogs and human CRBN in complex with human DDB1 and lenalidomide.
    Figure 1: The structures of thalidomide analogs and human ​CRBN in complex with human ​DDB1 and ​lenalidomide.

    (a) The chemical structure of ​thalidomide, with the ​glutarimide ring shown in red and the ​phthalimide moiety in black, along with an FoFc electron density difference map calculated in the absence of the molecule and contoured at 3σ. (b) As in a, showing ​pomalidomide. (c) As in a, showing ​lenalidomide with the ​isoindolinone moiety in black. (d) Structure of ​CRBN–​DDB1. The ​CRBN LLD is shown in yellow, the ​CRBN TBD in blue and the region deleted in a human polymorphism associated with mild mental retardation in red. ​DDB1 is shown in green with a gray surface, with β-propeller domains labeled BPA, BPB and BPC. ​Lenalidomide is shown as yellow sticks and the zinc ion as a gray sphere. N and C termini of ​Cereblon are labeled.

  2. Structural comparisons of CRBN–DDB1.
    Figure 2: Structural comparisons of ​CRBN–​DDB1.

    (a) Structural superposition of ​CRBN with B. subtilisLon protease (red, PDB 3M65). The ​CRBN LLD is shown in yellow and the TBD in blue. The ​DDB1-binding motif is inserted into the LLD. The TBD exhibits no similarity to ​Lon protease and occurs at the C terminus. (b) Structural superposition of ​CRBN–​DDB1 (yellow and green, respectively) with DDB–​DDB1 (​DDB2 shown in blue; ​DDB1 omitted). ​CRBN helices interacting with ​DDB1 are labeled A–E.

  3. Structure of the TBD of human and mouse CRBN.
    Figure 3: Structure of the TBD of human and mouse ​CRBN.

    (a) The human TBD is shown in blue, ​lenalidomide in yellow sticks and the LLD in yellow. Backbone atoms are represented only for residues 376–380 to show bonding. Three hydrogen bonds between the TBD and ​lenalidomide are shown as dashed lines to the protein backbone at His378 and Trp380 and also to the side chain of His378. The zinc-binding site is shown as a gray sphere and is ~18 Å from ​lenalidomide. The β-sheet region, which varies in the other structures, is marked by a β. (b) Mouse TBD in complex with ​thalidomide. (c) Mouse TBD in complex with ​pomalidomide.

  4. Sequence-alignment differences mapped onto the surface of the TBD of CRBN.
    Figure 4: Sequence-alignment differences mapped onto the surface of the TBD of ​CRBN.

    (a) Alignment with ​tryptophan residues forming the tri-Trp pocket shown in bold. Variable residues proximal to the tri-Trp pocket are shown in red. (b) Sequence alignment from a mapped onto the surface of the human ​CRBN TBD. Conserved residues relative to the human protein are shown in red, conservative changes in orange and nonconservative changes in white. The IMiD compound–binding pocket is indicated by a blue arrow.

  5. Conserved tryptophan residues (Trp386 and Trp400) confer binding of CRBN to IMiD compounds and are required for drug function in cells.
    Figure 5: Conserved ​tryptophan residues (Trp386 and Trp400) confer binding of ​CRBN to IMiD compounds and are required for drug function in cells.

    (a) Immunoblot (IB) analysis of extracts prepared from DF15R cells reexpressing full-length ​CRBN wild type or W386A or W400A mutants and preincubated with vehicle ​DMSO (V), ​thalidomide (​Thal) or ​pomalidomide (​Pom) at the indicated concentrations and bound to thalidomide-analog affinity beads, washed and eluted with SDS buffer. Input extract before bead purification (In) is also shown. Immunoblots are representative of three independent experiments. (b) Cell proliferation assay of DF15 cells (sensitive), DF15R cells (resistant; ​CRBNnull) and DF15R cells transduced with a red fluorescent protein (RFP) control construct, human (h) ​CRBNWT, ​CRBNW386A or ​CRBNW400A, treated with a dose response of ​pomalidomide for 5 d. Values are mean and s.d. from three independent experiments performed on different days. Data for each cell line are normalized to treatment with vehicle (​DMSO). (c) Immunoblot analysis of ​CRBN, ​Aiolos, ​c-Myc, ​IRF4, phospho-​Rb (pp​Rb) and ​β-tubulin loading control in DF15, DF15R, DF15R ​CRBNWT and DF15R ​CRBNW386A treated with vehicle ​DMSO (V), 10 μM ​lenalidomide (​Len) or 1 μM ​pomalidomide (​Pom) for 48 h.

  6. Mouse CRBN does not rescue pomalidomide effects in DF15R cells.
    Figure 6: Mouse ​CRBN does not rescue ​pomalidomide effects in DF15R cells.

    (a) Cell proliferation assay of DF15 (sensitive), DF15R (resistant; ​CRBNnull), DF15R-​hCRBNWT (human ​CRBN) and DF15R-​mCRBNWT (mouse ​CRBN) cells treated with a dose response of ​pomalidomide for 5 d. Values are mean and s.d. from three independent experiments performed on different days. Data for each cell line were normalized to treatment with vehicle (​DMSO). (b) ​CRBN immunoblot (IB) of extracts from DF15, DF15R and DF15R cells reexpressing full-length mouse ​CRBN wild type (​mCRBNWT). ​β-tubulin is a loading control. (c) ​Aiolos and ​Ikaros immunoblots of extracts from DF15 and DF15R cells reexpressing ​mCRBN treated with ​pomalidomide at the indicated concentrations for 48 h. ​β-tubulin is a loading control.

  7. CRBN binding assay with thalidomide enantiomers.
    Supplementary Fig. 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 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.

  8. The thalidomide-binding domain (TBD) of mouse Cereblon showing the crystal contacts formed between protein monomers, bridged by thalidomide molecules.
    Supplementary Fig. 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.

  9. Structural comparison between the TBD tri-Trp pockets.
    Supplementary Fig. 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.

  10. Structural superposition of Cereblon TBD with homologs.
    Supplementary Fig. 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.

  11. Immunoblot and immunohistochemical quantiification of CRBN.
    Supplementary Fig. 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.

  12. IL-2 co-stimulation by pomalidomide in human PBMCs but not in mouse splenocytes.
    Supplementary Fig. 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) Co-stimulation 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.

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Author information

Affiliations

  1. Celgene Corporation, San Diego, California, USA.

    • Philip P Chamberlain,
    • Antonia Lopez-Girona,
    • Karen Miller,
    • Gilles Carmel,
    • Barbra Pagarigan,
    • Barbara Chie-Leon,
    • Emily Rychak,
    • Laura G Corral,
    • Yan J Ren,
    • Maria Wang,
    • Mariko Riley,
    • Silvia L Delker,
    • Thomas O Daniel &
    • Brian E Cathers
  2. Tokyo Medical University, Tokyo, Japan.

    • Takumi Ito,
    • Hideki Ando &
    • Hiroshi Handa
  3. Nara Institute of Science and Technology, Nara, Japan.

    • Tomoyuki Mori,
    • Yoshinori Hirano &
    • Toshio Hakoshima

Contributions

P.P.C., G.C., B.P., B.C.-L., S.L.D., M.R., T.M., Y.H. and T.H. performed protein chemistry and structural studies. A.L.-G., K.M., E.R., L.G.C., Y.J.R., M.W., T.I. and H.A. performed biochemical and cellular experiments. P.P.C., A.L.-G., H.H., T.H., T.O.D. and B.E.C. planned the work, and all authors contributed to the manuscript.

Competing financial interests

P.P.C., A.L.-G., K.M., G.C., B.P., B.C.-L., E.R., L.G.C., Y.J.R., M.W., M.R., S.L.D., T.O.D. and B.E.C. are employees of Celgene. H.H. receives research support from Celgene.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: ​CRBN binding assay with thalidomide enantiomers. (307 KB)

    (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 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.

  2. Supplementary Figure 2: The thalidomide-binding domain (TBD) of mouse ​Cereblon showing the crystal contacts formed between protein monomers, bridged by ​thalidomide molecules. (309 KB)

    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. (281 KB)

    (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. (384 KB)

    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. (189 KB)

    (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. (63 KB)

    (a) Co-stimulation of ​IL-2 release by ​pomalidomide in human PBMC cells treated with anti-CD3. Data shown as means ±s.d.. (b) Co-stimulation 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.

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  2. Supplementary Data Set 1 (182 KB)

    Original images of gels and western blots used in this manuscript

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