DNA binding drives phase separation of the Gcn4 bZIP domain and reveals its conformational ensemble in the diluted and condensate phases

Liquid-liquid phase separation is widely invoked in transcriptional regulation, yet prevailing models attribute condensate formation primarily to intrinsically disordered activation domains rather than structured DNA-binding motifs. Here, we overturn this view by demonstrating that the isolated basic leucine zipper (bZIP) domain of the yeast transcription factor Gcn4 undergoes robust DNA-induced phase separation in the complete absence of its activation domain. Using small-angle X-ray scattering in combination with all-atom molecular dynamics simulations and ensemble optimization, we directly resolve the conformational landscape of the Gcn4 bZIP-DNA complex across coexisting dilute and condensed phases. Beyond the canonical uninterrupted helical conformation captured in crystal structures, we identify a previously unrecognized minor population featuring a pronounced helical kink at the basic region-leucine zipper junction. These findings establish DNA binding as a sufficient physical driver of bZIP phase separation and demonstrate that small-angle scattering can quantitatively interrogate protein conformational ensembles within biomolecular condensates, opening new avenues for the structural chemistry of phase-separated systems.