Differential chemical labeling of the AlcR DNA-binding domain from Aspergillas nidulans versus its complex with a 16-mer DNA target: Identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid

DNA binding of the ethanol regulon transcription factor AlcR from Aspergill us nidulans was shown to involve a consensus basic region as in the other z inc cluster proteins. However, additional interactions between some residue s and DNA were suspected, among which were a hypothetic hydrophobic interac tion between Trp45 and the T residue of the consensus TGCGG sequence. In th e present study, the differential chemical labeling of both the free protei n and the protein/DNA complex showed significantly different behaviors of t he three tryptophan residues comprised in the AlcR sequence toward the Kosh land reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface . A similar result obtained with Trp53, an amino acid present at the C-term inal side of AlcR, also indicated its involvement in the DNA recognition. I n contrast, the formation of the complex accompanied by an allosteric rearr angement allowed the Trp36 to be much more exposed to the solvent than in t he free protein. These data provide additional evidence that the unique spe cificity of AlcR among the zinc binuclear cluster family results in new typ es of interactions between AlcR and its cognate targets, From a methodologi cal point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the r ecognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.