Experimental identification of homodimerizing B-ZIP families in Homo sapiens.
B-ZIP transcription factors dimerization is mediated by a parallel coiled-coil termed the leucine zipper. We have evaluated the dimerization specificity of the seven coiled-coil B-ZIP proteins (ATF6, XBP, LZIP, NFIL3, TEF, CREB, and C/EBPalpha) with themselves and each other. To do this, we designed dominant negative proteins, termed A-ZIPs, that contain the leucine zipper dimerization domain of a B-ZIP protein and an acidic amphipathic N-terminal extension. The A-ZIPs heterodimerize with B-ZIP proteins in a leucine zipper-dependent manner. The acidic N-terminal extension is hypothesized to form an heterodimeric coiled-coil structure with the basic region, essentially zippering the leucine zipper into the basic region. We now present a new acidic extension design that stabilizes heterodimerization with B-ZIP proteins up to 11 kcal mol(-1). We have used these A-ZIP proteins in a competition EMSA to evaluate which A-ZIP can prevent DNA binding of which B-ZIP domain. Inhibition of DNA binding is interpreted to indicate that the A-ZIP is forming a heterodimer with the B-ZIP domain and thus prevents the B-ZIP from binding to DNA. All leucine zippers examined can homodimerize and two pairs (CREB & NFIL3 and ATF6 & XBP) can heterodimerize. We discuss these results with reference to the amino acid sequence of the leucine zipper region. These A-ZIP reagents may be of value in biological systems to inhibit the DNA binding and transcriptional potential of specific B-ZIP families.
Acharya
Asha
A
Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bldg. 37, Rm. 3128, Bethesda, MD 20892, USA.
Rishi
Vikas
V
Moll
Jonathan
J
Vinson
Charles
C
D051698:Activating Transcription Factor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051702:Activating Transcription Factor 6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism / Q000379:methods; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006358:Hot Temperature; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D051698:Activating Transcription Factor 2; D051702:Activating Transcription Factor 6; D050976:Basic-Leucine Zipper Transcription Factors; C497440:NFIL3 protein, human; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun