Interaction of the bHLH-zip domain of c-Myc with H1-type peptides. Characterization of helicity in the H1 peptides by NMR.

Abstract

The carboxyl-terminal 92 residues of c-Myc-92 display site-specific DNA binding specificity for the consensus sequence 5'-CACCACGTGGTG-3' (Blackwell, T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N., and Weintraub, H. (1990) Science 250, 1149-1151). Size exclusion high pressure liquid chromatography analysis of the purified fusion protein, glutathione S-transferase-c-Myc-92, indicates that c-Myc-92 is tetrameric with a dissociation constant of < 60 nM. Helix-1 (H1) and leucine zipper peptides from the basic-helix-loop-helix-zipper domain of c-Myc and Max were assayed as potential inhibitors of c-Myc-92 DNA binding. H1 peptides with substitutions that confer greater helicity are found to inhibit c-Myc-92 DNA binding. The mechanism of inhibition involves the cooperative binding of H1 peptides with tetrameric c-Myc-92 as determined by a spectrophotometric assay employing 2,4-dinitrophenyl-H1-F8A. NMR structural characterization reveals a correlation between helicity and inhibition. In a partially hydrophobic environment, H1-Mx (from Max) is a random coil, while H1-WT, H1-F8A, and H1-F8A,S6A (from c-Myc) display differing degrees of helicity. Structure determination on the basis of nuclear Overhauser effect data indicates that the H1-F8A helix is significantly more ordered than H1-WT. Analysis on the basis of the Max x-ray structure (Ferré-D'Amaré, R., Prendergast, G. C., Ziff, E. B., and Burley, S. K. (1993) Nature 363, 38-45) suggests that H1 peptide binding to c-Myc-92 may occur through an alteration in the packing of helix-1 in c-Myc-92 or through an interaction with an exposed hydrophobic cluster of residues at each H1-H2 interface. This binding site for H1 peptides may be of significance in the interaction of c-Myc with proteins involved in transcriptional regulation.