Biophysical Studies of the Interaction Between FOXO1 and the N-terminal Domain of the Androgen Receptor

Abstract

The androgen receptor (AR) drives prostate cancer progression. Resistance to ligand-binding domain (LBD)–targeted therapies highlights the importance of regulatory interactions within the intrinsically disordered N-terminal domain (NTD). A human transcription factor, Forkhead Box O1 (FOXO1), represses androgen- independent AR activity by binding the TAU-5 region. However, the underlying biophysical mechanism for this interaction remains unclear. Here, we investigate how FOXO1’s conserved forkhead domain and the subsequent unstructured nuclear localization sequence (FHDNLS) engage the Transcriptional Activation Unit-5 (TAU-5) within the AR NTD. Using Fluorescence Polarization (FP), multidimensional Nuclear Magnetic Resonance (NMR) spectroscopy, and Isothermal Titration Calorimetry (ITC), we show that FOXO1 FHDNLS binds the WXXLF motif, a transcriptional hot spot within TAU-5, with low-micromolar affinity. However, the extended TAU-5 region provides most of the binding interface and a stronger binding affinity than the minimal WXXLF motif. Additional docking simulation supports a weak, multivalent interface between FHDNLS and TAU-5. Confocal microscopy with Fluorescence Recovery After Photobleaching (FRAP) further reveals that FOXO1 FHDNLS and AR TAU- 5 co-partition into shared phase-separated droplets while maintaining distinct internal mobilities. This behavior is consistent with a dynamic and weakly associated complex. Together, these findings provide a biophysical framework for FOXO1-mediated repression of androgen-independent AR signaling within the context of the disordered NTD.