The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins (IDPs), mediates the receptor's transactivation function to regulate gene expression. However, an accurate molecular dissection of NTD's structure-function relationships remains elusive. Here, using small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), circular dichroism, and hydrogen exchange mass spectrometry, we show that NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion upon chemical denaturation. By combining SAXS, hydroxyl radical protein footprinting and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map that reveals metastable regional and long-range contacts, including interactions 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) NMR that mutations near residue I33 alter 19F chemical shifts at residue S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of IDPs' structure-function relationship, and how specific metastable contacts mediate critical functions of disordered proteins.
From Sequence and Forces to Structure, Function, and Evolution of Intrinsically Disordered Proteins
