Structure, function, and dynamics of the dimerization and DNA-binding domain of oncogenic transcription factor v-Myc

The protein product (c-Myc) of the protooncogene c-myc is a transcriptional regulator playing a key role in cellular growth, differentiation, and apop tosis. Deregulated myc genes, like the transduced retroviral v-myc allele, are oncogenic and cause cell transformation. The C-terminal bHLHZip domain of v-Myc, encompassing protein dimerization (helix-loop-helix, leucine zipp er) and DNA contact (basic region) surfaces, was expressed in bacteria as a highly soluble p15(v-myc) recombinant protein. Dissociation constants (K-d ) for the heterodimer formed with the recombinant bHLHZip domain of the Myc binding partner Max (p14(max)) and for the Myc-Max-DNA complex were estima ted using circular dichroism (CD) spectroscopy and quantitative electrophor etic mobility shift assay (EMSA). Multi-dimensional NMR spectroscopy was us ed to characterize the solution structural and dynamic properties of the v- Myc bHLHZip domain. Significant secondary chemical shifts indicate the pres ence of two separated a-helical regions. The C-terminal leucine zipper regi on forms a compact alpha -helix, while the N-terminal basic region exhibits conformational averaging with substantial. alpha -helical content. Both he lices lack stabilizing tertiary side-chain interactions and represent excep tional examples for loosely coupled secondary structural segments in a nati ve protein. These results and CD thermal denaturation data indicate a monom eric state of the v-Myc bHLHZip domain. The N-15 relaxation data revealed b ackbone mobilities which corroborate the existence of a partially folded st ate, and suggest a "beads-on-a-string'' motional behaviour of the v-Myc bHL HZip domain in solution. The preformation of alpha -helical regions was con firmed by CD thermal denaturation studies, and quantification of the entrop y changes caused by the hydrophobic effect and the reduction of conformatio nal entropy upon protein dimerization. The restricted conformational space of the v-Myc bHLHZip domain reduces the entropy penalty associated with het erodimerization and allows rapid and accurate recognition by the authentic Myc binding partner Max. (C) 2001 Academic Press.