UC San Diego

The NF-κB family of transcription factors is a central mediator of immune and inflammatory responses. Dysregulation of NF-κB results in numerous diseases including cancers. Immediate activation and deactivation of NF-κB through macromolecular interactions is crucial. This dissertation investigated the role of conformational dynamics in these interactions by combining single-molecule FRET (smFRET) and molecular dynamics (MD) simulations. All-atom MD simulations showed that three different NF-ĸB dimers, RelA–p50, the RelA homodimer, and the p50 homodimer, display distinct relative domain motions leading to different degrees of binding cavity exposure that correlate with experimental DNA-binding rates. These motions were directly observed in real-time by smFRET, revealing a heterogeneous conformational ensemble. Interconversion between conformational states and DNA-binding events occur on the same time scale, suggesting they are coupled. Remarkably, the motions were also observed when NF-κB was bound to DNA, inferring a dynamic picture unexpected from crystal structures. These motions were allosterically altered by the inhibitor protein IκBα, which is known to accelerate DNA-dissociation and prevent DNA-binding. Coarse-grained MD simulations were also performed to predict the conformational ensemble of the disordered transactivation domain of NF-ĸB and captured the helix propensity of the motifs that become structured when bound to the transcription coactivator CBP domains. Subunit exchange between NF-ĸB dimers was also investigated with simple chemical kinetic equations. Together, this dissertation provides a molecular understanding of the conformational dynamics in NF-ĸB transcriptional regulation and may contribute to the ultimate deciphering of the dynamics-function relationship of proteins.