Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation

The mammalian NF-κB p52:p52 homodimer together with its cofactor Bcl3 activates transcription of κB sites with a central G/C base pair (bp), while it is inactive toward κB sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined its structure in complex with a P-selectin(PSel)-κB DNA (5’-GGGGT<underline>G</underline>ACCCC-3’) (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-κB-DNA complex structures, which have a central A/T bp. Molecular dynamics (MD) simulations show free DNAs exist in distinct preferred conformations, and p52:p52 homodimer induces the least amount of conformational changes on the more transcriptionally active natural PSel-κB DNA in the bound form. Our binding assays further demonstrate that the fast kinetics driven by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for κB DNA in complex with NF-κB and suggest the importance of binding kinetics, dictated by free DNA conformational and dynamic states, in controlling transcriptional activation for NF-κB.