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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 acid¹

² Laboratoire de Spectrométrie de Masse, Institut de Chimie des Substances Naturelles, Centre National pour la Recherche Scientifique, F-91198 Gif sur Yvette Cedex, France
³ Laboratoire de Résonance Magnétique Nucléaire, Institut de Chimie des Substances Naturelles, Centre National pour la Recherche Scientifique, F-91198 Gif sur Yvette Cedex, France
⁴ Institut de Génétique et Microbiologie, UMR 8621, Centre Universitaire d'Orsay, Bâtiment 409, F-91405 Orsay Cedex, France

Reprint requests to: Olivier Laprévote, Laboratoire de Spectrométrie de Masse, Institut de Chimie des Substances Naturelles, Centre National pour la Recherche Scientifique, F-91198 Gif sur Yvette cedex; e-mail: laprevote{at}icsn.cnrs-gif.fr; fax: (33) 169 077 247.

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland 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-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

Keywords: AlcR; transcriptional factor; DNA-binding protein; Aspergillus nidulans; differential chemical modification; mass spectrometry

Abbreviations: av., average • calc., calculated • CM, carboxamidomethylated • deconv., deconvoluted • DNA, deoxyribonucleic acid • ds, double strand • DTT, DL-dithiothreitol • E, enzyme; EDTA, ethylenediaminetetraacetic acid • GST, glutathione-S-transferase • HPLC, high performance liquid chromatography • m/z, mass to charge ratio • MW, molecular weight • NMR, nuclear magnetic resonance • obs., observed • S, substrate • ss, single strand • TEAB, triethylammonium bicarbonate • Tris.HCl, tris(hydroxymethyl)aminomethane hydrochloride • TPCK, L-1-tosylamide-2-phenylethylchloromethyl ketone • v, volume • Vs, skimmer voltage • Vsc, sampling cone voltage • w, weight.

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