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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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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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.
All authors are employees of Novartis, or were at the time of this study.
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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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