Design and properties of a Myc derivative that efficiently homodimerizes.
bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and specific protein-protein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and off cell proliferation, differentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization specificity. We report that Myc recognition specificity is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding efficiency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.
Soucek
L
L
Università La Sapienza, Centro Acidi Nucleici CNR, Rome, Italy.
Helmer-Citterich
M
M
Sacco
A
A
Jucker
R
R
Cesareni
G
G
Nasi
S
S
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D016259:Genes, myc; D006131:Growth Inhibitors / Q000494:pharmacology; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D055503:Protein Multimerization; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014157:Transcription Factors
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; C499330:MAX protein, human; C067850:Myc associated factor X; D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C568271:omomyc protein; C068422:Max protein, mouse