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FC0042
Protein Max  -  DNA


Biological function
Max belongs to the family of b-HLH-LZ (helix-loop-helix, leucine zipper) transcription factors, which contains numerous proteins including the Myc and Mad families. Max recognizes and binds E-Box DNA sequences as a homodimer in vitro and represses transcription of Myc target genes in vivo.

Structural evidence
In its free form, Max dominantly exists as a homodimer, with the LZ domain adopting a helical structure. The LZ region, however, is unstable, and undergoes folding–unfolding transitions. This equilibrium s shifted towards the folded state by the N- and C-terminal segments, which remain as a random coil even in the dimer form. Biologically important regions flanking its bHLH-Zip motif are disordered or absent in crystal structures. These regions are resistant to proteolysis in both the presence and absence of DNA, and that Max dimers containing both flanking regions have significantly higher helix content as measured by circular dichroism than that predicted from the crystal structures. Owing to additional mobility in the ms to s time scale, the NMR data clearly indicate that only a limited part of the residues in Max1–132 can be disordered.

Biochemical evidence
The two major isoforms differ in the insertion N terminal to the basic region. The gene product p21 Max has a KD of 7 × 10-6, a value 10-100 times smaller than the b-HLH-LZ itself. This indicates that the disordered N- and C- terminal portions of p21 are involved in the stabilization of the homodimer.

Structure/Mechanism
The acidic N terminus masks the destabilizing electrostatic potential present at the LZ region. Owing to a strong cooperativity between the bHLH and LZ domains, the N- and C-terminal regions also facilitate the formation of the recognition helices in the bHLH region, thereby increasing the dissociation constant with the E-box by 10 to 100-fold.

Mechanism category
Conformational selection

Posttranslational modification
Phosphorylation of the Max transcription factor modulates the structure/disorder balance of the basic region of the DNA binding domain (DBD), thereby influencing its affinity for DNA. During Fas induced apoptosis the regions outside the b-HLH-LZ motif are first dephosphorylated and subsequently cleaved off by specific caspases. Phosphorylation regulates the helical content. The C and N terminal regions represent auto-inhibitory domains.

Isoforms, context-dependence
Synergistically regulated by two distinct mechanisms: phosphorylation and alternative splicing. The p22 Max isoform contains a nine-residue long insertion in the disordered N-terminal region, as compared to the shorter p21 isoform. The longer NTD is more effective in stabilizing the DBD and results in higher DNA binding affinity. Phosphorylation increases affinity in the long p22 isoform.

Significance
Fuzzy regions could modulate DNA binding affinity by modulating the helical content of b-HLH-LZ via an electrostatic mechanism.