Structural and thermodynamical characterization of the complete p21 gene product of Max.
The b-HLH-LZ family of transcription factors contains numerous proteins including the Myc and Mad families of proteins. Max heterodimerizes with other members to bind the E-Box DNA sequence in target gene promoters. Max is the only protein in this network that recognizes and binds E-Box DNA sequences as a homodimer in vitro and represses transcription of Myc target genes in vivo. Key information such as the structure of p21 Max, the complete gene product, and its KD in the absence of DNA are still unknown. Here, we report the characterization of the secondary and quaternary structures, the dimerization and DNA binding of p21 Max and a thermodynamically stable mutant. The helical content of p21 Max indicates that its N-terminal and C-terminal regions are unstructured in the absence of DNA. NMR experiments further support the location of folded and unfolded domains. We also show that p21 Max has an apparent KD (37 degrees C) of 7 x 10(-6), a value 10-100 times smaller than the b-HLH-LZ itself. We demonstrate that electrostatic repulsions are responsible for the higher KD of the b-HLH-LZ. Finally, we show that a p21 Max double mutant forms a very stable dimer with a KD (37 degrees C) of 3 x 10(-10) and that the protein/DNA complex depicts a higher temperature of denaturation than p21 Max/DNA complex. Our results indicate that Max could homodimerize, bind DNA, and repress transcription in vivo and that its mutant could be more efficient at repressing the expression of c-Myc target genes.
Naud
Jean-François
JF
Département de pharmacologie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4.
McDuff
François-Olivier
FO
Sauvé
Simon
S
Montagne
Martin
M
Webb
Bradley A
BA
Smith
Steven P
SP
Chabot
Benoit
B
Lavigne
Pierre
P
D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014461:Ultracentrifugation
D050976:Basic-Leucine Zipper Transcription Factors; C067850:Myc associated factor X