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

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...

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Published in:The Journal of biological chemistry Vol. 299; no. 7; p. 104855
Main Authors: Somsen, Bente A., Sijbesma, Eline, Leysen, Seppe, Honzejkova, Karolina, Visser, Emira J., Cossar, Peter J., Obšil, Tomáš, Brunsveld, Luc, Ottmann, Christian
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01.07.2023
American Society for Biochemistry and Molecular Biology
Subjects:
14-3-3 protein
14-3-3 Proteins - genetics
14-3-3 Proteins - metabolism
Drug Discovery
Estrogen Antagonists - pharmacology
Estrogen Receptor
Estrogen Receptor alpha - genetics
Estrogen Receptor alpha - metabolism
Humans
Ligands
Nuclear receptors
PPI stabilization
Protein Binding - drug effects
protein–protein interactions
Tamoxifen - pharmacology
protein–protein interactions
AE
ERα
Estrogen Receptor
DBD
PPI
GR
Nuclear receptors
E2
NRs
AUC
LBD
4-OHT
SEC
FC-A
14-3-3 protein
DSF
SV-AUC
PPI stabilization
FA
HDX
ISSN:0021-9258, 1083-351X, 1083-351X
Online Access:Get full text
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Summary: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.
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ISSN:0021-9258
1083-351X
1083-351X
DOI:10.1016/j.jbc.2023.104855