Role of Conformational Plasticity in Determining the DNA Binding Affinity of Different NFκB Dimers

Abstract

The Nuclear factor κappa B (NFκB) family proteins play a significant role in the regulation of genes associated with immune response, cell growth and development. The activation of NFκB family proteins is tightly regulated by the IκB inhibitor protein like IκBα and IκBβ in cells and its misregulation can lead to diseases such as cancer. There are five members of the NFκB family proteins: RelA (p65), RelB, c-Rel, p50, (NFKB1) and p52 (NFKB2). These proteins contain a conserved Rel Homology domain (RHD) responsible for DNA-binding and dimerization. Different NFκB protein homo or heterodimers work together to regulate distinct but overlapping set of genes. The NFκB dimers bind to 9 to 10 bp of DNA sites (κB sites) with different binding affinities. Much of the structural information available are from NFκBs either bound to DNA or an inhibitor IκB protein. To understand the dynamics of free NFκB proteins, we analyzed the free RelA homodimer and the p50/RelA heterodimer using hydrogen/deuterium exchange mass spectrometry. The results showed surprising differences in the dynamics of RelA in the homo- vs. heterodimer. We also performed all-atom molecular dynamics (MD) simulations to study the differences in the two. Both our H/D exchange and MD-simulation results provide evidence for an interaction between the DNA-binding domains in the homodimer that is absent in heterodimer. We surmised that this interaction would need to be broken for DNA binding to occur and that this might be the cause of the lower DNA binding affinity for the RelA homodimer as compared to the p50/RelA. These results are supported by our stopped flow experiments which showed a slower association rate for DNA binding to RelA homodimer. Further, these NFκBs showed distinct structural changes upon binding to the inhibitors.