Dimerization specificity of myogenic helix-loop-helix DNA-binding factors directed by nonconserved hydrophilic residues.
The myogenic regulatory factor MyoD dimerizes with other positive and negative regulatory factors through a conserved region called the helix-loop-helix (HLH) domain. Using a non-DNA-binding MyoD mutant with a normal HLH domain as a dimerization competitor in gel mobility shift assays in conjunction with various MyoD HLH mutants, nonhydrophobic amino acids were identified in the HLH domain that contribute to dimerization specificity with E12. The assay detected subtle differences in dimerization activity among the mutant MyoD proteins that correlated with their ability to activate transcription in vivo, but this correlation was not apparent in the absence of competitor. The identification of such nonhydrophobic residues enabled us to predict the differences in dimerization affinity among the four vertebrate myogenic factors with E12. The experiments confirmed the prediction. Furthermore, a high-affinity homodimerizing analog of MyoD was designed by a single substitution at one of these residue positions. These experimental results were strengthened when they were analyzed in terms of the crystal structure for the Max bHLHZip domain homodimer. This analysis has allowed us to identify those residues that form charged residue pairs between the two HLH domains of MyoD and E12 and determine the dimerization specificity of the bHLH proteins.
Shirakata
M
M
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Friedman
F K
FK
Wei
Q
Q
Paterson
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001704:Biopolymers / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000502:physiology
D001704:Biopolymers; D004268:DNA-Binding Proteins; D017570:MyoD Protein; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D010649:Phenylalanine