Our ability to respond to a myriad of external stressors is dependent on the precise activity of specific transcription factors. The nuclear factor κB (NFκB) transcription factor family and especially its most abundant member—the p50p65 heterodimer—is key to our cellular inflammatory response. The heterodimer’s two subunits, p50 and p65, interact with each other through dimerization domains that connect by flexible linkers to N-terminal domains (NTDs), all together forming an extensive DNA binding pocket mediated by both base-specific contacts and nonspecific electrostatic interactions with the DNA backbone. The NTDs of p50 and p65 interact with DNA at two separate ends of a 10-base pair consensus binding sequence (5’-GGGRNNYYCC-3’). While these consensus binding sites are prevalent in our genomes, partial consensus sites or “half sites” are more common, increasing the number of potential p50p65 targets. Here, using hydrogen deuterium exchange mass spectrometry and molecular dynamics simulations to investigate protein structural dynamics, we observe that nucleotide substitution at the p65-interacting end of a consensus DNA sequence to form a half-site induces dynamic structural changes at the linkers and NTDs of both the p65 and p50 subunits. Our results show that despite this coordinated NTD conformational rearrangement on half-site DNA, p50p65 is capable of binding half-site DNA with high affinity due to its multifaceted DNA interactions. We also explore the surprising role of the middle, “nonspecific” nucleotides of the consensus sequence in subtly tuning binding affinity. Our results help to explain how p50p65 binds a wide range of target DNA sequences with high affinity and can therefore activate transcription of hundreds of genes.
Gunther et al. (Sun,) studied this question.