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Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex

Nature Chemical Biology volume 16, pages 15–23 (2020)Cite this article

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Abstract

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15–RBM39 interaction, we solved the DCAF15–DDB1–DDA1–indisulam–RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.

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Fig. 1: Functional validation of RBM39 and DCAF15–DDB1–DDA1 complexes.
Fig. 2: Structural analysis of the human DCAF15–DDB1–DDA1–RBM39(RRM2) complex with indisulam.
Fig. 3: DDA1 stabilizes the DCAF15–DDB1 complex and impacts degradation of RBM39 by indisulam.
Fig. 4: Detailed description of indisulam binding at the DCAF15 and RBM39 interface.
Fig. 5: Proteome-wide motif search predicts indisulam selectivity confirmed by expression proteomics.

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Data availability

The authors declare that the data supporting the findings of this study are available within the publication and its Supplementary Information or have been deposited in the PDB or Electron Microscopy Data Bank (http://www.ebi.ac.uk/pdbe/emdb/), as appropriate. Further information is available upon request. The PDB accession code for the human DCAF15–DDB1–DDA1–RBM39(RRM2)–indisulam EM co-structure is 6SJ7 and the EMDB accession code is EMD-10213. The PDB accession codes for the X-ray co-strutures of human DCAF15–DDB1(ΔBPB)–DDA1–RBM39(RRM2)–indisulam and human DCAF15–DDB1(ΔBPB)–DDA1–RBM39(RRM2)–compound 9 are 6UD7 and 6UE5, respectively.

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Acknowledgements

The authors thank M. Renatus (Novartis) for providing DCAF15 constructs and M. Li (Novartis) for providing DDB1 and DDB1(ΔBPB) constructs. We also thank G. Pardee for baculovirus generation and protein expression, S. Widger for additional expression support, X. Ma for helpful discussions on ligase structural biology, S. Skolnik for supporting pKa measurements, and T. Rejtar for help with proteomics data informatics. We would like to thank T. Terwilliger and R. Read for the helpful discussions in regards to crystallographic molecular replacement and phasing. Finally, we thank J. Bradner, J. Shulok, R. Jain, J. Porter and J. Tallarico for helpful discussions, input on this manuscript and supporting this work.

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  1. These authors jointly supervised this work: Jonathan Solomon, Joshiawa Paulk.

Authors and Affiliations

  1. Novartis Institutes for Biomedical Research, Emeryville, CA, USA

    Dirksen E. Bussiere, Lili Xie, Wei Shu, Andreas O. Frank, Alexandra Frommlet & Mark Knapp

  2. Novartis Institutes for Biomedical Research, Basel, Switzerland

    Honnappa Srinivas, Celine Be, Nathalie Carte, Patrick Graff, Hans Voshol & Christian Wiesmann

  3. Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    Ashley Burke, Junping Zhao, Adarsh Godbole, Dan King, Rajeshri G. Karki, Viktor Hornak, Fangmin Xu, Jennifer Cobb, Aleem Fazal, Rohan Beckwith, Jonathan M. Solomon & Joshiawa Paulk

  4. Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA

    Barun Okram, Songchun Jiang & Pierre-Yves Michellys

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Contributions

R.B., A. Fazal, J.Z., B.O., S.J. and P.-Y.M. designed and/or synthesized reported compounds. N.C., P.G. and H.V. performed crosslinking and mass spectrometry studies. A.B., D.K. and A.G performed SPR experiments. D.K. performed analytical ultracentrifugation. V.H. and R.G.K. performed proteome-wide motif searches and structural and computational modeling. C.B., H.S. and C.W. collected and processed cryo-EM data. F.X. and J.C. conducted expression proteomics experiments. A.O.F. and A. Frommlet performed biological NMR experiments. W.S. performed crystallographic screening and crystal optimization; D.E.B. designed protein constructs, collected X-ray crystallography datasets, reduced data, determined initial crystal structures and refined final structures; M.K. refined the final structures. L.X. purified DCAF15 complexes and RBM39 variants, and performed ITC and DSF experiments. J.P. and A.B. purified RBM39 variants, performed fluorescence-polarization and TR-FRET assays, cellular viability assays, siRNA knockdown and immunoblots. All authors contributed to writing. D.E.B., J.M.S., and J.P wrote and edited the final manuscript. D.E.B., J.M.S., L.X., and J.P contributed intellectual and strategic input.

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Correspondence to Dirksen E. Bussiere, Jonathan M. Solomon or Joshiawa Paulk.

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Supplementary Tables 1 and 2 and Supplementary Figs. 1–16

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Bussiere, D.E., Xie, L., Srinivas, H. et al. Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex. Nat Chem Biol 16, 15–23 (2020). https://doi.org/10.1038/s41589-019-0411-6

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