title: Structural basis for DNA recognition by the basic region leucine zipper transcription factor CCAAT/enhancer-binding protein alpha.
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.
authors.affiliation: Protein Structure Section, Macromolecular Crystallography Laboratory, and the Regulation of Cell Growth Laboratory, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201, USA. millerm@ncifcrf.gov
authors.forename: Maria
authors.initials: M
authors.lastname: Miller
authors.forename: Jon D
authors.initials: JD
authors.lastname: Shuman
authors.forename: Thomas
authors.initials: T
authors.lastname: Sebastian
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Dauter
authors.forename: Peter F
authors.initials: PF
authors.lastname: Johnson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024361:Amino Acids, Basic; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D016350:Leucine Zippers / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D051381:Rats; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D024361:Amino Acids, Basic; D022763:CCAAT-Enhancer-Binding Protein-alpha; D014157:Transcription Factors; D004247:DNA