title: Crystal structure of the minimalist Max-E47 protein chimera.
abstract: Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5'-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47 dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Faraz
authors.initials: F
authors.lastname: Ahmadpour
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.forename: Antonia T
authors.initials: AT
authors.lastname: De Jong
authors.forename: Melanie
authors.initials: M
authors.lastname: Gloyd
authors.forename: Jumi A
authors.initials: JA
authors.lastname: Shin
authors.forename: Alba
authors.initials: A
authors.lastname: Guarné
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D017510:Protein Folding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058127:Transcription Factor 3 / Q000235:genetics / Q000378:metabolism*
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; C067850:Myc associated factor X; D011993:Recombinant Fusion Proteins; D058127:Transcription Factor 3