Crystal Structure of the Minimalist Max-E47 Protein Chimera

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.

Combining rational design and continuous evolution on minimalist proteins that target DNA

“…Mutating Cys29 to Ala greatly improved protein tractability in in vitro assays. In our ME47 crystal structure, 13 the sulfur atoms in the Cys29 residues from the two monomers are 10.6 Å away from one another; given that typical disulfide distances are 2-3 Å, 60 we did not expect Cys dimerization to occur.…”

“…3). 13 Ser29. This double mutant also performed poorly, particularly in the B1H, and displayed fairly weak helical structure by CD ( Fig.…”

“…5 ME47 comprises 66 amino acids (aa) and is a hybrid of the DNA-binding basic region from Max, a bHLHZ protein, and the HLH dimerization domain from E47, a bHLH protein. 5,12,13 Another protein, MFW (84 aa, previously termed "MaxFosW" 14 ), is a modified Max bHLHZ in which the native Max leucine zipper (LZ) is replaced by FosW, 15,16 which is a designed LZ variant based on the bZIP protein c-Fos. [17][18][19] Interestingly, the engineered FosW homodimerizes, while the native c-Fos LZ does not.…”

“…Our work demonstrates the clear advantages of combining our rational design know-how with the unpredicted, yet beneficial, mutations uncovered via PACE to design the more structurally consistent and robust minimalist protein MEF with improved E-box binding function. 13 ) and FosW (PDB: 5FV8 51 ) crystal structures were merged to illustrate the intended structure of the MEF dimer; monomers highlighted in light and dark blue (Chimera v. 1.11.2 52 ). B. DNA sequences used in reporter constructs for B1H, CD, and EMSA.…”

Peptide therapeutics that directly target transcription factors

“…Hence, ME47's mechanism of action is believed to be as it was originally designed: to competitively inhibit MYC/MAX binding to the E‐box, which is demonstrated by ME47's high‐affinity, sequence‐specific recognition of the E‐box target ( K d 15.3 nM) as well as its E‐box‐binding profile in the ChIP experiments . The crystal structure demonstrates that ME47 homodimerizes through its E47 HLH . ME47 does not interact with MYC, as shown by yeast two‐hybrid (Y2H) assay, and therefore, it is unlikely that the effects observed in the MDA‐MB‐231 cancer cells resulted from ME47 interfering with the protein‐protein interaction of the MYC/MAX heterodimer.…”