Identification of a novel determinant for basic domain-leucine zipper DNA binding activity in the acute-phase inducible nuclear factor-interleukin-6 transcription factor.
Nuclear factor-interleukin-6 (NF-IL6), a member of the CCAAT box/enhancer-binding protein (C/EBP) family, contains a basic domain-leucine zipper (bZIP) DNA binding motif. Controlled protease digestion was used to probe free and DNA-complexed NF-IL6 protein. Digestion with trypsin in the absence of DNA produced the leucine zipper domain (containing residues 303-345). In contrast, digestion of NF-IL6.DNA complexes produced a stable domain, spanning residues 266-345, termed the tryptic core domain (TCD). The NH2-terminal boundary of the TCD is longer than tryptic peptides reported from C/EBP alpha.DNA complexes. Digestion of NF-IL6 with endoprotease Asp-N produced a domain smaller than the TCD (NF-IL6 bZIP domains (NFBD) (272-345)), a domain identified either in the absence or the presence of DNA. Both recombinant peptides bind acute-phase response element DNA in a sequence-specific fashion. The equilibrium disassociation constant (Kd) for the TCD was 36 +/- 8 nM, whereas the Kd for NFBD (272-345) was 283 +/- 160 nM. Moreover, in comparison with the TCD, NFBD (272-345) formed unstable DNA complexes with a 15-fold faster off-rate. We conclude that the amino acids represented between 266 and 272 termed the complex stabilizing subdomain, influences DNA complex formation independent of DNA binding specificity, and may be one mechanism for heterogeneity of DNA interaction by C/EBP family members.
Brasier
A R
AR
Department of Medicine, University of Texas Medical Branch, Galveston 77555-1060.
Kumar
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D015850:Interleukin-6 / Q000378:metabolism*; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D009687:Nuclear Proteins; D014157:Transcription Factors; D004247:DNA; D014357:Trypsin