A Metastable Contact and Structural Disorder in the Estrogen Receptor Transactivation Domain

The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins, mediates the receptor's transactivation function. However, an accurate molecular dissection of NTD's structure-function relationships remains elusive. Here, we show that the NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion on chemical denaturation. By combining small-angle X-ray scattering, hydroxyl radical protein footprinting, and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map revealing metastable long-range contacts, e.g., between residues I33 and S118. We show that mutation at S118, a known phosphorylation site, promotes conformational changes and increases coactivator binding. We further demonstrate via fluorine-19 (19F) nuclear magnetic resonance that mutations near I33 alter 19F chemical shifts at S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of how specific contact metastability mediates critical functions of disordered proteins. [Display omitted] •A compact disorder is observed for the transactivation domain of estrogen receptor•Multi-technique data integration determines ensemble-structures of protein disorder•Contact map uncovers metastable, long-range contacts between Ile33 and Ser118•19F NMR validates the role of contact metastability in receptor function We unraveled that the transactivation domain of estrogen receptor is structurally disordered, yet unexpectedly compact, and that a metastable Ile33-Ser118 contact is observed to inhibit coactivator binding. Disruption by mutagenesis alters ensemble-structures and coactivator binding, providing a functional link of oncogenic Ser118 phosphorylation in breast cancer endocrine resistance.