MyoD-E12 heterodimers and MyoD-MyoD homodimers are equally stable.
Muscle development is controlled by the MyoD family of basic helix-loop-helix (bHLH) DNA-binding proteins. These proteins dimerize with ubiquitous products of the E2A gene (E12 and E47) and bind in a sequence-specific manner to enhancer regions of muscle-specific genes activating their expression. In this study, fluorescence anisotropy has been utilized to characterize the interactions of recombinant MyoD and E12 in solution in the absence of DNA. The Gibb's free energies of dissociation (deltaG) and the equilibrium dissociation constants (K(D)) for the protein-protein interactions are reported. The deltaG for the MyoD homodimers in 100 mM KCl was 8.7 kcal/mol (K(D) = 340 nM), and increasing the salt concentration resulted in destabilization of the dimer. From titrations of MyoD-dansyl with E12 at 100 mM KCl, a free energy of heterodimerization of 8.7 (+0.4/-2.4) kcal/mol was recovered using rigorous confidence limit testing. The titrations of E12-dansyl with MyoD yielded a free energy of 8.3 kcal/mol with tighter confidence limits, +0.5/-0.8 kcal/mol. Thus, in the absence of DNA, both MyoD homodimers and MyoD-E12 heterodimers are relatively weak complexes of approximately the same stability. E12 does not form stable homo-oligomeric complexes; remaining monomeric at concentrations as high as 20 microM. Based on these results and the apparent binding constants reported previously for DNA binding, DNA is likely to facilitate the dimerization of MyoD and E12. Furthermore, higher affinity interactions of MyoD-E12 heterodimers versus MyoD homodimers with DNA binding sites is not due to preferential heterodimerization.
Maleki
S J
SJ
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Royer
C A
CA
Hurlburt
B K
BK
D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004355:Drug Stability; D005454:Fluorescence Polarization; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051379:Mice; D017570:MyoD Protein / Q000737:chemistry* / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D051820:TCF Transcription Factors; D013816:Thermodynamics; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D017570:MyoD Protein; D051820:TCF Transcription Factors; C544912:TCF7L1 protein, human; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D011189:Potassium Chloride; D004247:DNA