MON-267 Influence of Flanking Sequences Within the N-terminal Domain on the Structure and Functions of the Glucocorticoid Receptor’s AF1

Abstract Disclosure: S.H. Khan: None. R. Kumar: None. Most of our understanding of the two transcriptional activation function (AF) domains of glucocorticoid receptor (GR) is with AF2 located within the globular ligand binding domain (LBD). By contrast, little is known about AF1 that resides within the N-terminal domain (NTD), including its structure, the biologically relevant interacting coregulators and mechanism of activation. This deficiency is due, in part, to the fact that the NTD/AF1 is composed largely of intrinsically disordered polypeptide (IDP) and is not amenable to traditional high resolution structure analysis by NMR and crystallography. However, IDPs undergo a disorder-order transition upon interaction with target proteins by a “coupled folding and binding process” that can result in stabilization of an active structural conformation. We have earlier shown that TATA-Box Binding Protein (TBP) can induce folding of the GR AF1 domain in a manner that facilitates binding of Steroid Receptor Coactivator-1 (SRC-1) and correlates with AF1 activity. However, the molecular and structural basis for these interactions and mechanism of AF1 activation remains poorly defined. In the study, we tested whether the flanking sequences around AF1 affect its conformation and subsequent AF1-mediated GR activity. Using biophysical techniques, we found that placing AF1 immediately upstream from the DNA binding domain (DBD) leads to formation of a relatively ordered conformation in ID AF1, which is further stabilized via coupled folding and binding transition when encountered by TBP. Our analyses also revealed that removing amino acid sequences prior to AF1 or removal of amino acids between AF1 and DBD differentially regulates AF1-mediated GR activity via AF1’s interaction with SRC-1. The results from this study may provide an explanation for the cell/tissue-specific GR selectivity. Presentation: Monday, July 14, 2025