Molecular basis and dual ligand regulation of tetrameric Estrogen Receptor α/14-3-3ζ protein complex

Bente A. Somsen, Eline Sijbesma, Seppe Leysen, Karolina Honzejkova, Emira J. Visser, Peter J. Cossar, Tomáš Obšil, Luc Brunsveld (Corresponding author), Christian Ottmann (Corresponding author)

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Abstract

Therapeutic strategies targeting Nuclear Receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest, driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the Estrogen Receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα, however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its Ligand Binding Domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.

Original languageEnglish
Article number104855
Number of pages16
JournalJournal of Biological Chemistry
Volume299
Issue number7
DOIs
Publication statusPublished - Jul 2023

Bibliographical note

Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Funding

We would like to thank Inga Tharun and Anniek den Hamer for their initial work on ERα protein expression and native chemical ligation efforts. We would like to thank Sebastian van den Wildenberg and Sylvia Rovers-Genet for their help with the high-resolution MS experiments, Petr Pompach and Pavla Vaňková for their work on the HDX experiments and data processing, and Suzanne Timmermans for her help on the analytical SEC experiments. Furthermore, we acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Crystallography data collection was carried out at PETRA III using beam P11. Beamtime was allocated for proposal I-20200853 EC. Finally, we like to acknowledge Veronika Obšilová for her guidance and insight into the project. B. A. S. E. S. S. L. E. J. V. L. B. and C. O. conceptualization; B. A. S, E. S, S. L. and K. H investigation; B. A. S. and K. H. formal analysis; T. O, P. J. C. L. B. and C. O. supervision; P. J. C. L. B. and C. O. project administration; B. A. S. visualization; B. A. S. writing–original draft; B. A. S. E. S. S. L. E. J. V. L. B. K. H. T. O, P. J. C. and C. O. writing–review & editing; B. A. S. T. O. L. B. and C. O. funding acquisition. The research described was funded by The Netherlands Organization for Scientific Research via NWO Echo grant 711.017.014 and Gravity Program 024.001.035. Furthermore, this work benefited from access to the Native Mass Spectrometry Facility of Biocev. The financial support was provided by Instruct-ERIC (PID: 21877). The research described was funded by The Netherlands Organization for Scientific Research via NWO Echo grant 711.017.014 and Gravity Program 024.001.035 . Furthermore, this work benefited from access to the Native Mass Spectrometry Facility of Biocev. The financial support was provided by Instruct-ERIC (PID: 21877 ).

FundersFunder number
Nederlandse Organisatie voor Wetenschappelijk Onderzoek711.017.014, 024.001.035, 21877
Helmholtz Association

    Keywords

    • 14-3-3 protein
    • Estrogen Receptor
    • Nuclear receptors
    • PPI stabilization
    • protein–protein interactions

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