IDENTIFICATION OF A NOVEL DETERMINANT FOR BASIC DOMAIN-LEUCINE ZIPPERDNA-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/enhan cer-binding protein (C/EBP) family, contains a basic domain leucine zi pper (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 prod uced a stable domain, spanning residues 266-345, termed the tryptic co re domain (TCD). The NH2-terminal boundary of the TCD is longer than t ryptic 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 acu te-phase response element DNA in a sequence-specific fashion. The equi librium disassociation constant (K-d) for the TCD was 36 +/- nM, where as the K-d for NFBD (272-345) was 283 +/- 160 nM. Moreover, in compari son 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, influenc es DNA complex formation independent of DNA binding specificity, and m ay be one mechanism for heterogeneity of DNA interaction by C/EBP fami ly members.