Domain-Specific EPR Spectroscopy to Monitor Facilitated Dissociation of a DNA-Transcription Factor Complex

Transcription factors (TFs) regulate gene expression by interacting with specific DNA sequences in various manners, yet the mechanisms by which they dissociate from high-affinity DNA sites under physiological conditions remain not completely understood. Here, we employ continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy in native solution state to resolve, with domain-level precision, the dissociation dynamics of the MYC-associated factor X (MAX) from its cognate EBOX DNA motif. Site-directed spin labeling reveals that in the absence of DNA, MAX undergoes a stepwise dissociation process—beginning with melting of the N-terminal disordered region (NTD), followed by the helix-loop-helix (HLH) domain, and culminating in leucine zipper (LZ) dimer dissociation. DNA binding reorganizes this process into a cooperative all-or-none transition, wherein destabilization of the LZ triggers concerted collapse of the entire trimeric MAX:MAX–DNA complex. Strikingly, the addition of a disordered BRCA1 fragment (residues 219–504) disrupts this cooperativity by selectively destabilizing DNA contacts in the NTD and HLH regions, without perturbing the LZ. This results in facilitated dissociation via competitive DNA binding, observed here directly at domain-level resolution. Our findings establish EPR spectroscopy as a uniquely sensitive tool for dissecting TF–DNA dynamics under physiological conditions, offering mechanistic insight into regulated unbinding processes inaccessible to ensemble methods.