New structural determinants for c-Myc specific heterodimerization with Max and development of a novel homodimeric c-Myc b-HLH-LZ.
c-Myc must heterodimerize with Max to accomplish its functions as a transcription factor. This specific heterodimerization occurs through the b-HLH-LZ (basic region, helix 1-loop-helix 2-leucine zipper) domains. In fact, many studies have shown that the c-Myc b-HLH-LZ (c-Myc'SH) preferentially forms a heterodimer with the Max b-HLH-LZ (Max'SH). The primary mechanism underlying the specific heterodimerization lies on the destabilization of both homodimers and the formation of a more stable heterodimer. In this regard, it has been widely reported that c-Myc'SH has low solubility and homodimerizes poorly and that repulsions within the LZ domain account for the homodimer instability. Here, we show that replacing one residue in the basic region and one residue in Helix 1 (H(1)) of c-Myc'SH with corresponding residues conserved in b-HLH proteins confers to c-Myc'SH a higher propensity to form a stable homodimer in solution. In stark contrast to the wild-type protein, this double mutant (L362R, R367L) of the c-Myc b-HLH-LZ (c-Myc'RL) shows limited heterodimerization with Max'SH in vitro. In addition, c-Myc'RL forms highly stable and soluble complexes with canonical as well as non-canonical E-box probes. Altogether, our results demonstrate for the first time that structural determinants driving the specific heterodimerization of c-Myc and Max are embedded in the basic region and H(1) of c-Myc and that these can be exploited to engineer a novel homodimeric c-Myc b-HLH-LZ with the ability of binding the E-box sequence autonomously and with high affinity.
Département de Pharmacologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Québec, J1H 5N4, Canada.
Marie-Eve
ME
Beaulieu
François-Olivier
FO
McDuff
Vincent
V
Frappier
Martin
M
Montagne
Jean-François
JF
Naud
Pierre
P
Lavigne
D000595:Amino Acid Sequence; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018257:Helix-Loop-Helix Motifs / Q000235:genetics / Q000502:physiology; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology*; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology*; D020836:Protein Structure, Quaternary; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C499330:MAX protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc