Improving the thermodynamic stability of the leucine zipper of max increases the stability of its b-HLH-LZ:E-box complex.
Max is a member of the b-HLH-LZ (basic region-helix1-loop-helix2-leucine zipper) family of eukaryotic transcription factors. It is the obligate partner of the related b-HLH-LZ proteins, c-Myc and Mad1, with which it forms heterodimers on target DNA. While c-Myc and Mad1 require Max for DNA-binding, Max itself can form a homodimer that recognizes E-box DNA sequences (CACGTG) in gene promoters that are targeted by c-Myc. Evidence suggests that this mode of binding by Max may repress c-Myc transcriptional activity, and this may have applications in the control of the aberrant activity of c-Myc during certain oncogenic transformations. To enhance this repressive potential of Max, we sought to stabilize Max homodimers. We have designed a double mutant (N78V/H81L) located in the coiled-coil interface of the leucine zipper domain and we demonstrate that these mutations do indeed increase the stability of the protein. The mutations also improve the stability of the complex with cognate DNA. Thermal denaturations monitored by circular dichroism reveal two transitions that are due to intermediate folding states for both the wild-type and mutant proteins; this is supported by detailed thermodynamic analyses. A formalism to characterize the temperature-dependence of the unfolding, including the effect of intermediates, is presented.
Jean-François
Naud
N
Département de Microbiologie et Infectiologie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4.
Frédéric
Gagnon
G
Raymund
Wellinger
W
Benoit
Chabot
C
Lavigne
Pierre
P
D000595:Amino Acid Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D024721:E-Box Elements; D018257:Helix-Loop-Helix Motifs; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D011995:Recombination, Genetic; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D017931:DNA Primers; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C067850:Myc associated factor X; D014157:Transcription Factors; D004247:DNA