Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15

Article metrics

Abstract

The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1–DCAF15–DDA1 core ligase complex bound to RBM39 and E7820 at a resolution of 4.4 Å, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a new fold stabilized by DDA1, and that extensive protein–protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrate how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high-affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Cryo-EM structure of the DDB1∆B–DCAF15–DDA1 complex bound to E7820 and RBM39RRM2.
Fig. 2: Crystal structure of the DDB1∆B–DCAF15split–DDA1–E7820–RBM39RRM2 complex.
Fig. 3: DDA1 stabilizes the CRL4DCAF15 complex and facilitates RBM39 recruitment.
Fig. 4: Aryl-sulfonamide binding to DCAF15.
Fig. 5: Interprotein contacts between DCAF15 and RBM39.
Fig. 6: Topological and evolutionary constraints on E7820 activity.

Data availability

Structural coordinates for DDB1∆B–DDA1–DCAF15–E7820–RBM39, DDB1∆B–DDA1–DCAF15–tasisulam–RBM39 and DDB1∆B–DDA1–DCAF15–indisulam–RBM39 have been deposited in the PDB under accession numbers 6Q0R, 6Q0V and 6Q0W. The cryo-EM volume data are available at the Electron Microscopy Data Bank under accession numbers EMD-20554 and EMD-20553. MS raw data files have been deposited in the Proteomics Identifications archive under accession number PXD014536. Other data and materials are available from the authors upon reasonable request.

References

  1. 1.

    Salami, J. & Crews, C. M. Waste disposal—an attractive strategy for cancer therapy. Science 355, 1163–1167 (2017).

  2. 2.

    Chamberlain, P. P. et al. Structure of the human Cereblon–DDB1–lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat. Struct. Mol. Biol. 21, 803–809 (2014).

  3. 3.

    Fischer, E. S., Park, E., Eck, M. J. & Thoma, N. H. SPLINTS: small-molecule protein ligand interface stabilizers. Curr. Opin. Struct. Biol. 37, 115–122 (2016).

  4. 4.

    Fischer, E. S. et al. Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014).

  5. 5.

    Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350 (2010).

  6. 6.

    Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014).

  7. 7.

    Kronke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).

  8. 8.

    Gandhi, A. K. et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4CRBN. Br. J. Haematol. 164, 811–821 (2014).

  9. 9.

    Donovan, K. A. et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. Elife 7, e38430 (2018).

  10. 10.

    Sievers, Q. L. et al. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362, eaat0572 (2018).

  11. 11.

    Sheard, L. B. et al. Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468, 400–405 (2010).

  12. 12.

    Tan, X. et al. Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446, 640–645 (2007).

  13. 13.

    Uehara, T. et al. Selective degradation of splicing factor CAPERα by anticancer sulfonamides. Nat. Chem. Biol. 13, 675–680 (2017).

  14. 14.

    Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356, eaal3755 (2017).

  15. 15.

    Ozawa, Y. et al. E7070, a novel sulphonamide agent with potent antitumour activity in vitro and in vivo. Eur. J. Cancer 37, 2275–2282 (2001).

  16. 16.

    Wang, E. et al. Targeting an RNA-binding protein network in acute myeloid leukemia. Cancer Cell 35, 369–384 (2019). e367.

  17. 17.

    Assi, R. et al. Final results of a phase 2, open-label study of indisulam, idarubicin, and cytarabine in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome. Cancer 124, 2758–2765 (2018).

  18. 18.

    Petzold, G., Fischer, E. S. & Thoma, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4 ubiquitin ligase. Nature 532, 127–130 (2016).

  19. 19.

    Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D Biol. Crystallogr. 71, 136–153 (2015).

  20. 20.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  21. 21.

    Fischer, E. S. et al. The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation. Cell 147, 1024–1039 (2011).

  22. 22.

    Zimmerman, E. S., . & Schulman, B. A. & Zheng, N. Structural assembly of cullin-RING ubiquitin ligase complexes. Curr. Opin. Struct. Biol. 20, 714–721 (2010).

  23. 23.

    Jin, J., Arias, E. E., Chen, J., Harper, J. W. & Walter, J. C. A family of diverse Cul4–Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol. Cell 23, 709–721 (2006).

  24. 24.

    Shabek, N. et al. Structural insights into DDA1 function as a core component of the CRL4–DDB1 ubiquitin ligase. Cell Disco. 4, 67 (2018).

  25. 25.

    Olma, M. H. et al. An interaction network of the mammalian COP9 signalosome identifies Dda1 as a core subunit of multiple Cul4-based E3 ligases. J. Cell Sci. 122, 1035–1044 (2009).

  26. 26.

    Cavadini, S. et al. Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome. Nature 531, 598–603 (2016).

  27. 27.

    Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535, 252–257 (2016).

  28. 28.

    Nowak, R. P. et al. Plasticity in binding confers selectivity in ligand-induced protein degradation. Nat. Chem. Biol. 14, 706–714 (2018).

  29. 29.

    Landau, M. et al. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, W299–W302 (2005).

  30. 30.

    Abdulrahman, W. et al. A set of baculovirus transfer vectors for screening of affinity tags and parallel expression strategies. Anal. Biochem. 385, 383–385 (2009).

  31. 31.

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

  32. 32.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

  33. 33.

    Mccoy, A. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  34. 34.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  35. 35.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

  36. 36.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

  37. 37.

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

  38. 38.

    Moriya, T. et al. High-resolution single particle analysis from electron cryo-microscopy images using SPHIRE. J. Vis. Exp. 123, e55448 (2017).

  39. 39.

    Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166 (2018).

  40. 40.

    Link, A. J. & LaBaer, J. Trichloroacetic acid (TCA) precipitation of proteins. Cold Spring Harb. Protoc. 2011, 993–994 (2011).

  41. 41.

    Gundry, R. L. et al. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr. Protoc. Mol. Biol. Chapter 10, Unit10.25 (2009).

Download references

Acknowledgements

We acknowledge H.-S. Seo for help with ITC experiments. Cryo-EM data were collected at the UMass cryo-EM facility, with help from K. Song and C. Xu. Financial support for this work was provided by NIH grant NCI R01CA214608 (to E.S.F.) and an F32 fellowship 1F32CA232772-01 (to T.F.). E.S.F. is a Damon Runyon-Rachleff Innovator supported in part by the Damon Runyon Cancer Research Foundation (DRR-50–18). This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by NIH NIGMS (P41 GM103403) and NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated by Argonne National Laboratory under contract number DE-AC02-06CH11357. This research was, in part, supported by the National Cancer Institute’s National cryo-EM facility at the Frederick National Laboratory for Cancer Research.

Author information

T.B.F., H.Y., N.S.G., R.P.N. and E.S.F. initiated the project and designed experiments; T.B.F., H.Y. and R.P.N. conducted protein purification; T.B.F. performed crystallization and cryo-EM experiments; H.Y. developed and performed biochemical assays; R.P.N., T.B.F. and E.S.F. collected and processed X-ray diffraction data; N.A.E. and K.A.D. performed the MS experiments; H.Y., Z.L. and Q.C. synthesized small molecules with input from T.Z; N.S.G. and E.S.F. supervised the project; and T.B.F., H.Y. and E.S.F. wrote the manuscript with input from all authors.

Correspondence to Eric S. Fischer.

Ethics declarations

Competing interests

E.S.F. is a founder and/or member of the scientific advisory board (SAB) and equity holder of C4 Therapeutics and Civetta Therapeutics, and a consultant to Novartis, AbbVie and Pfizer. The Fischer laboratory receives research funding from Novartis, Deerfield and Astellas. N.S.G. is a founder, scientific advisory board member and equity holder in Gatekeeper, Syros, Petra, C4, B2S and Soltego. The Gray laboratory receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi. N.S.G., E.S.F, H.Y., Q.C., T.Z., T.F., R.P.N and K.A.D. are inventors on patent applications (PCT/US2018/065701 and PCT/US2019/014919) submitted by the Dana-Farber Cancer Institute.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Supplementary Figs. 1–6

Reporting Summary

Supplementary Note

Synthetic procedures

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Faust, T.B., Yoon, H., Nowak, R.P. et al. Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15. Nat Chem Biol (2019) doi:10.1038/s41589-019-0378-3

Download citation