Interaction of the bHLH-zip domain of c-Myc with H1-type peptides. Characterization of helicity in the H1 peptides by NMR.
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.
Draeger
L J
LJ
Department of Chemistry, University of Wisconsin, Milwaukee 53211.
Mullen
G P
GP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D016350:Leucine Zippers; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D004247:DNA; D005982:Glutathione Transferase