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.
Highlights
The basic helix-loop-helix proteins are a widely distributed superfamily of transcription factors that regulate genes important for cell proliferation, differentiation and apoptosis [1]
Despite the absence of its cognate DNA target, the overall structure of Max-E47 is virtually identical to those of other basic helix-loop-helix (bHLH) and bHLHZ dimers [13,15,16,38]. This finding was somewhat unexpected because it had been previously shown that addition of polyions, such as DNA, assists dimerization and increases the structural organization of bHLH and bHLHZ proteins [39]
The conformation of helix a1 is different from that of other bHLHZ structures determined in the presence of DNA (Figure 1)
Summary
The basic helix-loop-helix (bHLH) proteins are a widely distributed superfamily of transcription factors that regulate genes important for cell proliferation, differentiation and apoptosis [1]. These transcription factors comprise an N-terminal basic region (b) necessary for binding to a shared signature DNA-motif (59CANNTG) and a C-terminal helix-loop-helix (HLH) region that mediates homo- or heterodimerization [2,3]. Activation by Myc requires heterodimerization with Max, a bHLHZ transcription factor that serves to regulate other members of this superfamily [5,6,7]. Max forms heterodimers with other bHLHZ proteins, including the Mad transcription factor. The Mad1/Max complex functions as a transcriptional repressor [8,9,10,11] and, it has been suggested that Myc/Max and Mad/Max complexes define a molecular switch regulating the cellular transition from a growth to a resting state
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