Structural basis for DNA recognition by the basic region leucine zipper transcription factor CCAAT/enhancer-binding protein α

Abstract

CCAAT/enhancer-binding proteins (C/EBPs) are basic region leucine zipper (bZIP) transcription factors that regulate cell differentiation, growth, survival, and inflammation. To understand the molecular basis of DNA recognition by the C/EBP family we determined the x-ray structure of a C/EBPα bZIP polypeptide bound to its cognate DNA site (A-5T-4T-3G-2 C-1G1C2A3A4 T5) and characterized several basic region mutants. Binding specificity is provided by interactions of basic region residues Arg289, Asn292, Ala295, Val296, Ser299, and Arg300 with DNA bases. A striking feature of the C/EBPα protein-DNA interface that distinguishes it from known bZIP-DNA complexes is the central role of Arg289, which is hydrogen-bonded to base A3, phosphate, Asn292 (in-variant in bZIPs), and Asa293. The conformation of Arg289 is also restricted by Tyr285. In accordance with the structural model, mutation of Arg289 or a pair of its interacting partners (Tyr285 and Asn293) abolished C/EBPα binding activity. Val296 (Ala in most other bZIPs) contributes to C/EBPα specificity by discriminating against purines at position -3 and imposing steric restraints on the invariant Arg300. Mutating Val296 to Ala strongly enhanced C/EBPα binding to cAMP response element (CRE) sites while retaining affinity for C/EBP sites. Thus, Arg289 is essential for formation of the complementary protein-DNA interface, whereas Val296 functions primarily to restrict interactions with related sequences such as CRE sites rather than specifying binding to C/EBP sites. Our studies also help to explain the phenotypes of mice carrying targeted mutations in the C/EBPα 2bZIP region.