Crystal Structure of a Free κB DNA: Insights into DNA Recognition by Transcription Factor NF-κB Original Research Article

The dimeric NF-κB transcription factors regulate gene expression by recognizing specific DNA sequences located within the promoters of target genes. The DNA sequences, referred to as κB DNA, are divided into two broad classes. Class I κB DNA binds optimally to p50 and p52 NF-κB subunits, while class II κB DNAs are recognized specifically by the NF-κB subunits c-Rel and p65. We determined the X-ray crystal structure of a class II κB DNA sequence at 1.60 Å resolution. This structure provides a detailed picture of κB DNA hydration, counter ion binding, and conformation in the absence of NF-κB binding partner. X-ray structures of both class I and class II κB DNA bound to NF-κB dimers were determined previously. Additionally, the NMR solution structure of a class I κB DNA is known. Comparison of the protein-bound and unbound κB DNA structures reveals that the free form of both classes approximates ideal B-form DNA more closely. Local geometries about specific DNA bases differ significantly upon binding to NF-κB. This is particularly evident at the 5′-GG/CC base-pairs; a signature of NF-κB specific DNA binding sequences. Differential phosphate group conformations, minor groove widths, buckle, twist, and tilt angles are observed between bound and unbound κB DNA. We observe that the presence of an extra G:C base-pair, 5′- to the GGA sequence in class I κB DNA, alters the geometry of the two internal G:C base-pairs within the GGGA tetranucleotide, which explains, at least in part, the structural basis for distinct NF-κB dimer recruitment by the two different classes of κB DNA. Together, these observations suggest that NF-κB dimers recognize specific structural features of κB DNA in order to make sequence-specific complexes.