Addition of positively charged tripeptide to N-terminus of the Fos basic region leucine zipper domain: implications on DNA bending, affinity, and specificity.
GKH-Fos(139-211)/Jun(248-334) (GKH: glycine-lysine-histidine) is a modified Fos/Jun heterodimer designed to contain a metal binding motif in the form of a GKH tripeptide at the amino terminus of Fos bZIP domain dimerized with the Jun basic region leucine zipper (bZIP) domain. We examined the effect of the addition of positively charged GKH motif to the N-terminus of Fos(139-211) on the DNA binding characteristics of the Fos(139-211)/Jun(248-334) heterodimer. Binding studies indicate that while the nonspecific DNA binding affinity of the GKH modified heterodimer increases 4-fold, it specifically binds the activating protein-1 (AP-1) site 6-fold less tightly than the control unmodified counterpart. Furthermore, helical phasing analysis indicates that GKH-Fos(139-211)/Jun(248-334) and control Fos(139-211)/Jun(248-334) both bend the DNA at the AP-1 site toward the minor groove. However, due to the presence of the positively charged GKH motif on Fos, the degree of the induced bend by GKH- Fos(139-211)/Jun(248-334) is greater than that induced by the unmodified Fos/Jun heterodimer. Our results suggest that the unfavorable energetic cost of the increased DNA bending by GKH-Fos(139-211)/Jun(248-334) results in a decrease in both specificity and affinity of binding of the heterodimer to the AP-1 site. These findings may have important implications in protein design as well in our understanding of DNA bending and factors responsible for the functional specificity of different members of the bZIP family of transcription factors.
Department of Structural Biology and Biochemistry, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada.
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Mahmoudi
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Sarkar
D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D051790:G-Box Binding Factors; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry*; D016759:Oncogene Protein p65(gag-jun) / Q000737:chemistry; D016761:Oncogene Proteins v-fos / Q000737:chemistry*; D016415:Sequence Alignment; D018808:Transcription Factor AP-1 / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D016759:Oncogene Protein p65(gag-jun); D016761:Oncogene Proteins v-fos; D018808:Transcription Factor AP-1; D014157:Transcription Factors