Application of surface plasmon resonance to elucidate the cooperative DNA binding mechanisms of NFATc1 and c-Jun

Background: 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 cJun 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). Results: 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 f 0.07) & times; 10- 7 M, while c-Jun shows a slightly stronger affinity KD = (1.95 f 0.03) & times; 10- 7 M. Not surprisingly, when forming a heterodimeric complex, the NFATc1-c-Jun binding affinity further lowers the KD = (1.63 f 0.17) & times; 10- 7 M, indicating cooperative interaction. More important, kinetic analysis revealed that the association rate (ka) increased more than threefold, from (2.44 f 0.10) & times; 105 M- 1 s-1 to (8.29 f 0.19) & times; 105 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. Significance: 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.