DNA-mediated Folding and Assembly of MyoD-E47 Heterodimers

Abstract

and ¶Institut fü r Polymere, ETH Zü rich, Switzerland Basic region helix-loop-helix (bHLH) transcription factors regulate key steps in early development by binding to regulatory DNA sites as heterodimers consisting of a tissue-specific factor and a widely expressed factor. We have examined the folding, dimerization, and DNA binding properties of the muscle-specific bHLH protein MyoD and its partner E47, to understand why these proteins preferentially associate in heterodimeric complexes with DNA. In the absence of DNA, the E47 bHLH domain forms a very stable homodimer, whereas MyoD is unfolded and monomeric. Fluorescence quenching experiments show that MyoD does not dimerize with E47 under dilute conditions in the absence of DNA. Residues in and around the loop of the E47 bHLH domain contribute to its markedly greater stability. An altered MyoD bHLH substituted with the loop segment from E47 folds in the absence of DNA, and it readily dimerizes with E47. In the presence of a specific DNA binding site, MyoD and E47 both form homodimeric complexes with DNA that have similar dissociation constants, despite the very different stabilities of these protein dimers off DNA. A 1:1 mixture of these bHLH domains forms almost exclusively heterodimeric complexes on DNA. Assembly of these bHLH-DNA complexes is apparently governed by the strength of each subunit's interaction with the DNA and not by the strength of protein-protein interactions at the dimer interface. These findings suggest that preferential association of MyoD with E47 in DNA complexes results from more favorable DNA contacts made by one or both subunits of the heterodimer in comparison with either homodimeric complex.At the heart of transcriptional initiation are numerous cooperative interactions between basal factors, transcriptional activators, and their promoter DNA sites. Many activating transcription factors bind to their DNA targets as heterodimers in which each subunit has a distinctive DNA binding preference and transcriptional activity. The strength of transcriptional activation is modulated by the choice of subunits forming these protein dimers, their cellular concentrations, and their affinities for DNA regulatory sites. Basic region helix-loop-helix (bHLH)

Molecular Recognition in Helix-Loop-Helix and Helix-Loop-Helix-Leucine Zipper Domains

“…The presence of a number of residues that were found at high frequency in the HLH domains (Gln 22 and Thr 23 ; Ile 1 , Leu 5 , Met 11 /Val 11 , and Cys 12 ) did not correlate to either greater affinity or specificity to any of the targets, suggesting that these residues have a role in proper folding of the domain and its display on phage coat. The strong bias for the two loop residues Gln 22 and Thr 23 is in agreement with previous work describing the loop as a key determinant of bHLH stability (33). This role is particularly evident for Gln 22 , which occurred in all domains; its structural role is visible in the E47 dimer structure, where it participates, together with Gln 13 and Gln 30 , in a hydrogen bond network that connects the loop with helices 1 and 2, stabilizing the four helix bundle (19).…”

“…Thus, they appear to destabilize the dimers. Previous work suggested that heterodimers of MyoD with the E12 E-protein are stabilized by attractive pairs formed by Glu 3 , Glu 7 , and Glu 39 residues of E12 with MyoD residues Arg 29 , Arg 33 , and Gln 39 , respectively (34). Because more stable dimers can be obtained with noncharged amino acids, it seems that the role of the charged Glu residues in the E-protein is to prevent an excessively strong interaction with MyoD or Id2, allowing the physiological partner exchange.…”

“…Likewise, the amino acids in many Zip region positions (2, 8, 11, 12, 16, 23, 25, and 26) showed the same preference for Rox or Mad binding, indicating that these positions per se are unable to determine specificity. Actually, it was shown previously that it is necessary to mutate four residues (residues 5, 12, 18, and 19) in the Myc Zip to overcome its inability to dimerize (6), that Id1 dimerization specificity can be conferred to E47 by replacing four amino acids at the helix 1/loop junction (36), and that a 6-fold increase in MyoD bHLH dimer stability is obtained by substituting 18 amino acids from the loop and the adjacent regions of E47 (33). Most of the mutants identified as binders show affinity for more than one protein.…”

A recurrent dominant negative E47 mutation causes agammaglobulinemia and BCR– B cells

“…11), which encode both the DNA-binding domain, in the basic region, and the dimerization domain in the HLH region (12). Both E12 and E47 bind the consensus sequence CANNTG as a homodimer or heterodimer with other bHLH proteins, including tissue-specific master genes, such as myogenic differentiation (MyoD) (13).…”

Id Proteins Negatively Regulate Basic Helix-Loop-Helix Transcription Factor Function by Disrupting Subnuclear Compartmentalization

“…bHLH-bHLH dimer interactions are strongly stabilized as a ternary complex by DNA binding (43)(44)(45). In order to efficiently sequester bHLH proteins as Id-bHLH heterodimers, Id proteins must be able to overcome the large DNA-binding free energy of bHLH proteins.…”