Abstract
The main nuclear factor kappa B transcription factor family members RelA-p50 heterodimer and RelA homodimer have different biological functions and show different transcriptional activation profiles. To investigate whether the two family members adopt a similar conformation in their free states, we performed hydrogen-deuterium exchange mass spectrometry, all-atom molecular dynamics simulations, and stopped-flow binding kinetics experiments. Surprisingly, the N-terminal DNA-binding domains adopt an open conformation in RelA-p50 but a closed conformation in RelA homodimer. Both hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations indicate the formation of an interface between the N-terminal DNA-binding domains only in the RelA homodimer. Such an interface would be expected to impede DNA binding, and stopped-flow binding kinetics show that association of DNA is slower for the homodimer as compared to the heterodimer. Our results show that the DNA-binding cavity in the RelA-p50 heterodimer is open for DNA binding, whereas in the RelA homodimer, it is occluded.