Covalently linking BHLH subunits of MASH-1 increases specificity of DNA binding.

MASH-1, a member of the basic-helix-loop-helix (BHLH) family of transcription factors, promotes the differentiation of committed neuronal precursor cells. In vitro, MASH-1 displays only marginal DNA sequence specificity. We have produced a MASH-1 variant, MASH-GGC, by introducing the tripeptide Gly-Gly-Cys at the C-terminal end of the BHLH domain. Under reducing conditions the properties of MASH-GGC and of the BHLH domain of MASH-1 were very similar. Like MASH-1, reduced MASH-GGC showed little specificity of DNA binding. CD spectroscopy revealed that both proteins underwent a conformational change from a largely unfolded to a mainly alpha-helical conformation upon binding to DNA. When the subunits of MASH-GGC were linked through a disulfide bond, the folded conformation was stable over a wide concentration range (2.5 nM to 2 microM) even in the absence of DNA. Oxidized MASH-GGC bound to E-box-containing sequences half-maximally at 148 nM, compared to 458 nM for the reduced form. Therefore, even when the change from a monomeric to a dimeric species was taken into account, the affinity for E-box-containing DNA sequences was increased. Surprisingly, the apparent dissociation constant for the complex with DNA not containing E-box sequences was increased upon oxidation. Therefore, despite the large distance between the disulfide bridge and the protein-DNA interface, covalently linking the subunits of MASH-1 increased the specificity of DNA binding significantly. In vivo, such an increase of the intrinsic DNA binding specificity might be achieved through interactions with other proteins of the transcriptional machinery.