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/EBPalpha bZIP polypeptide bound to its cognate DNA site (A(-5)T(-4)T(-3)G(-2)C(-1)G(1)C(2)A(3)A(4)T(5)) and characterized several basic region mutants. Binding specificity is provided by interactions of basic region residues Arg(289), Asn(292), Ala(295), Val(296), Ser(299), and Arg(300) with DNA bases. A striking feature of the C/EBPalpha protein-DNA interface that distinguishes it from known bZIP-DNA complexes is the central role of Arg(289), which is hydrogen-bonded to base A(3), phosphate, Asn(292) (invariant in bZIPs), and Asn(293). The conformation of Arg(289) is also restricted by Tyr(285). In accordance with the structural model, mutation of Arg(289) or a pair of its interacting partners (Tyr(285) and Asn(293)) abolished C/EBPalpha binding activity. Val(296) (Ala in most other bZIPs) contributes to C/EBPalpha specificity by discriminating against purines at position -3 and imposing steric restraints on the invariant Arg(300). Mutating Val(296) to Ala strongly enhanced C/EBPalpha binding to cAMP response element (CRE) sites while retaining affinity for C/EBP sites. Thus, Arg(289) is essential for formation of the complementary protein-DNA interface, whereas Val(296) 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/EBPalpha bZIP region.
Highlights
CCAAT/enhancer-binding proteins (C/EBPs) are basic region leucine zipper transcription factors that regulate cell differentiation, growth, survival, and inflammation
X-ray structures of basic region leucine zipper (bZIP) domain peptides bound to DNA have been determined for GCN4 (bound either to an AP-1 site [18] or to a cAMP response element (CRE) site [19, 20]), the Jun/Fos heterodimer [21], CREB [22], and PAP1 [23]
Overview of the Complex—We crystallized a C/EBP␣ bZIP polypeptide bound to a 21-mer DNA duplex and determined the structure at 2.8 Å resolution (Fig. 2)
Summary
CCAAT/enhancer-binding proteins (C/EBPs) are basic region leucine zipper (bZIP) transcription factors that regulate cell differentiation, growth, survival, and inflammation. X-ray structures of bZIP domain peptides bound to DNA have been determined for GCN4 (bound either to an AP-1 site [18] or to a CRE site [19, 20]), the Jun/Fos heterodimer [21], CREB [22], and PAP1 [23] These structures show that bZIPs recognize specific DNA sites through base contacts made by five residues within the basic region motif characteristic for each subfamily. These five positions are well conserved among all bZIP proteins and include the invariant Asn and Arg residues (Fig. 1). The bZIP structures have revealed functional variability of conserved residues in DNA recognition
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