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.
Künne
A G
AG
Department of Chemistry, ETH-Zürich, Switzerland.
Allemann
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D018257:Helix-Loop-Helix Motifs; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; C065500:ASCL1 protein, human