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Structural basis of PROTAC cooperative recognition for selective protein degradation

Nature Chemical Biology volume 13, pages 514–521 (2017)Cite this article

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Abstract

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase–PROTAC–target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4BD2). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4BD2. Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.

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Figure 1: The crystal structure of the Brd4BD2–MZ1–VHL–ElonginC–ElonginB complex.
Figure 2: Brd4BD2 and VHL form a stable, cooperative complex in the presence of MZ1.
Figure 3: The molecular basis of MZ1-induced compact complex formation between Brd4BD2 and VHL.
Figure 4: Structure-guided design and characterization of Brd4-selective degrader AT1.
Figure 5: Schematic model of selective PROTAC-induced target degradation.

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Acknowledgements

This work was supported by the European Research Council (ERC-2012-StG-311460 DrugE3CRLs Starting Grant to A.C.); the UK Biotechnology and Biological Sciences Research Council (BBSRC grant BB/J001201/2 to A.C.); the European Commission (H2020-MSCA-IF-2014-655516 Marie Skłodowska-Curie Actions Individual Fellowship to K.-H.C. and H2020-MSCA-IF-2015-806323 Marie Skłodowska-Curie Actions Individual Fellowship to X.L.); and the Wellcome Trust (Strategic Awards 100476/Z/12/Z for biophysics and drug discovery and 094090/Z/10/Z for structural biology and X-ray crystallography to the Division of Biological Chemistry and Drug Discovery). We are thankful to P. Fyfe for support with the in-house X-ray facility; L. Finn for support with tissue culture facility (MRC-PPU); the Ferguson lab for access to LI-COR equipment; T. Cardote (Ciulli lab, BCDD, SLS, Dundee) for the gift of full-length Cul2-Rbx1 and A. Knebel (MRC-PPU/DSTT) for the gift of E1 and E2 enzymes; the Division of Computational Biology for support with computational cluster; and to Diamond Light Source for beamtime (BAG proposal MX10071) and beamline support at beamline I04-1.

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  1. Morgan S Gadd, Andrea Testa and Xavier Lucas: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, UK

    Morgan S Gadd, Andrea Testa, Xavier Lucas, Kwok-Ho Chan, Wenzhang Chen, Douglas J Lamont, Michael Zengerle & Alessio Ciulli

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Contributions

A.C. conceived the idea and directed the project. M.S.G., X.L., A.T., K.-H.C. and A.C. designed the experiments and interpreted results. M.S.G., X.L., A.T., and K.-H.C. performed experiments. A.T. and M.Z. contributed to compound design and synthesized compounds. W.C. performed MS proteomics experiments under the supervision of D.J.L. M.S.G., X.L. and A.C. wrote the manuscript with input from all other authors.

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Correspondence to Alessio Ciulli.

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Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–12 (PDF 4451 kb)

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Chemistry (PDF 2033 kb)

Supplementary Data Set

Proteomic analysis of relative protein abundance in HeLa cells. Results are graphically represented in Figure 4f,g (XLSX 7377 kb)

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Gadd, M., Testa, A., Lucas, X. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13, 514–521 (2017). https://doi.org/10.1038/nchembio.2329

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