Structural and Thermodynamical Characterization of the Complete p21 Gene Product of Max

Abstract

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.

New structural determinants for c‐Myc specific heterodimerization with Max and development of a novel homodimeric c‐Myc b‐HLH‐LZ,

“…As expected, the addition of the E‐box probe significantly increased the helical content of c‐Myc'RL (Figure a). Indeed, as reported elsewhere for other b‐HLH‐LZ (as well as for b‐LZ (Chan et al ., ) and b‐HLH (Cave et al ., ; Ferré‐D'amaré et al ., ; Fieber et al ., ; Montagne et al ., ; Naud et al ., ; Naud et al ., ; Rishi and Vinson, ; Wendt et al ., )), this increase comes from the stabilization of the b‐region into an α‐helix upon interaction with DNA (Cave et al ., ; Ferré‐D'amaré et al ., ; Fieber et al ., ; Montagne et al ., ; Naud et al ., ; Naud et al ., ; Rishi and Vinson, ; Wendt et al ., ). The [Θ] 222 nm of c‐Myc'RL at 20 °C in the presence of E‐box DNA is −7500 deg·cm 2 ·dmol –1 , which corresponds to ~21% of the maximal helix content expected for a peptide of this length (i.e., –36 000 deg·cm 2 ·dmol –1 as calculated from the classical formalism developed by Chen et al, ).…”

The Mechanism of Discrimination between Cognate and Non-Specific DNA by Dimeric b/HLH/LZ Transcription Factors

“…MaxVL is composed of residues A2-E83 of the p21 gene product with the N58V and H61L mutations in the LZ domain to optimize thermodynamical stability and NMR signal of the dimeric state. 25 A linker of four residues (GSGC) at the C terminus was added for the formation of a disulfide bond to prevent dissociation 22,25,29 (Figure 2(a)). The specific double-stranded DNA (SD) sequence was designed to contain the E-Box in the center element (CACGTG).…”

Classification of Intrinsically Disordered Regions and Proteins

“…The fuzzy N-terminal tail of the Max transcription factor, for example, reduces electrostatic repulsion in the basic helix–loop–helix (bHLH) domain and thereby facilitates formation of the DNA recognition helices, which increases binding affinity by 10–100-fold. 252 (ii) In the second mechanism, the disordered region(s) modulate flexibility of the binding interface. The serine- and arginine-rich region of the Ets-1 transcription factor exemplifies this mechanism, which reduces DNA binding affinity by 100–1000-fold.…”