Arginine (348) is a major determinant of the DNA binding specificity of transcription factor E12.

The basic helix-loop-helix proteins (BHLH) E12 and E47 bind to DNA in a cell-type specific fashion as heterodimers with transcription factors such as MyoD, Myf-5, MRF-4, myogenin, and MASH-1 and -2 which are critical regulators of cellular differentiation. We have measured the apparent dissociation constants (KD) of the complexes of E12 and several E12 mutants with various oligonucleotides. Glutamate (345) of E12, which is hydrogen bonded to a CpA dinucleotide, and arginine (348), a residue that does not directly interact with the nucleobases, are major determinants of the DNA binding specificity of E12. R(348) is in direct contact with both the phosphate backbone and the carboxylate of E(345), thereby locking the side chain conformation of E(345). In its locked conformation the glutamate residue interacts favourably only with E-box containing DNA, the natural target of BHLH-proteins, while repulsive interactions destabilise the complexes with all other DNA sequences.