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 xray structure of a C/EBPα_bZIP polypeptide bound to its cognate DNA site (A_5T_4T_3G_2C_1G1C2A3A4T5) 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 (invariant in bZIPs), and Asn293. 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_ bZIP region.

C/EBPs蛋白家族是亮氨酸拉链转录因子（bZIP）家族中的一个亚族，为了研究C/EBPs蛋白识别DNA的基本原理，本文选择C/EBPα入手，首先通过X射晶体线衍射法得到C/EBPα蛋白与DNA相互作用的三维结构，对晶体结构作进一步分析后对C/EBPα蛋白设计突变实验，提出C/EBPα蛋白识别DNA的模型。此模型可以促进C/EBPα蛋白家族功能的进一步研究。对于生物信息学家，可以在此家族蛋白质识别DNA模型的基础上利用统计学方法对转录因子的结合位点进行预测和分析。