Transcription factors (TFs) regulate gene expression and coordinate key cellular processes, including proliferation, differentiation, and immune responses. NFATc1 is a central regulator of immune signaling, while c-Jun mediates stress and oncogenic pathways. Their cooperative DNA binding is critical for controlling complex transcriptional programs, yet existing approaches inadequately capture the real-time kinetics underlying these interactions. This study addresses the lack of dynamic characterization of cooperative NFATc1 and c-Jun DNA binding using surface plasmon resonance (SPR). Using SPR, we quantified the individual and cooperative DNA-binding kinetics of NFATc1 and c-Jun. NFATc1 binds DNA with a dissociation constant (KD) of (4.11 ± 0.07) × 10 −7 M, while c-Jun shows a slightly stronger affinity KD = (1.95 ± 0.03) × 10 −7 M. Not surprisingly, when forming a heterodimeric complex, the NFATc1–c-Jun binding affinity further lowers the KD = (1.63 ± 0.17) × 10 −7 M, indicating cooperative interaction. More important, kinetic analysis revealed that the association rate (ka) increased more than threefold, from (2.44 ± 0.10) × 10 5 M −1 s −1 to (8.29 ± 0.19) × 10 5 M −1 s −1 , while dissociation kinetics remained dynamic. These results demonstrate that NFATc1 facilitates c-Jun recruitment, enhancing cooperative DNA engagement. Together, the findings highlight the unique ability of SPR to resolve cooperative TF-DNA interactions with high temporal precision, providing insights not attainable through conventional techniques. This study reveals a kinetic mechanism underlying NFATc1–c-Jun synergistic gene regulation and demonstrates the power of SPR to resolve cooperative TF–DNA interactions in real time, bridging static structural data with dynamic transcriptional regulation. • Quantified NFATc1 and c-Jun DNA binding kinetics using SPR • Revealed cooperative DNA binding in NFATc1–c-Jun heterodimer • Heterodimer formation increases binding affinity and association rate • SPR demonstrates enhanced sensitivity to TF-TF and TF-DNA interactions • Findings offer insights into transcription factor-targeted therapeutic design
Cao et al. (2026) studied this question.