Myc protein structure: localization of DNA-binding and protein dimerization domains.

ABSTRACT

Wildtype and mutant v-Myc proteins were overexpressed in Escherichia coli using the T7 RNA polymerase system, and the in vitro DNA-binding activities of partially or highly purified proteins were analysed by native DNA-cellulose chromatography. For the construction of the expression plasmids, cloned proviral DNA from wildtype MC29 or from its spontaneous deletion mutant Q10C was used, the latter lacking internal v-myc sequences. Both the wildtype (p59) and the mutant (p42) recombinant protein contain at their amino termini 12 amino acids encoded by the vector, followed by 11 gag amino acids and 9 amino acids encoded by v-myc sequences derived from noncoding c-myc sequences. In addition, p59 contains 416 amino acids encoded by v-myc sequences derived from the complete chicken c-myc coding region, whereas p42 lacks 120 amino acids from the central region of the Myc protein including the highly acidic domain. Two additional proteins were engineered which contain the first 309 (p53) or the last 107 (p16) amino acids, respectively, of the Myc protein sequence in addition to vector-encoded amino acids. The p16 protein represents the carboxyl terminus of the Myc protein sequence containing both a muscle determination gene (MyoD1) homology region, including a basic motif and an amphipathic helix-loop-helix motif, and a leucine heptad repeat. All proteins, except p53 which lacks the carboxyl-terminal Myc protein sequences, bound to native DNA-cellulose and were eluted with 200-500 mM NaCl. Based on the DNA-binding activities of recombinant or spontaneous mutant v-Myc proteins extracted from bacterial or from transformed avian cells, we conclude that the DNA-binding domain of avian Myc proteins is confined within the last 86 carboxyl-terminal amino acids. The same region is also shown to be necessary and sufficient for Myc protein dimerization. This 86-amino acid region essentially encompasses a putative basic DNA contact surface and a tandem array of two presumed protein dimerization motifs, helix-loop-helix and leucine repeat.