title: Toward the elucidation of the structural determinants responsible for the molecular recognition between Mad1 and Max.
abstract: Mad1 is a member of the Mad family. This family is part of the larger Myc/Max/Mad b-HLH-LZ eukaryotic transcription-factor network. Mad1 forms a specific heterodimer with Max and acts as a transcriptional repressor when bound to an E-box sequence (CACGTG) found in the promoter of c-Myc target genes. Mad1 cannot form a complex with DNA by itself under physiological conditions. A global model for the molecular recognition has emerged in which the Mad1 b-HLH-LZ homodimer is destabilized and the Mad/Max b-HLH-LZ heterodimer is favored. The detailed structural determinants responsible for the molecular recognition remain largely unknown. In this study, we focus on the elucidation of the structural determinants responsible for the destabilization of the Mad1 b-HLH-LZ homodimer. Conserved acidic residues at the dimerization interface (position a) of the LZ of all Max-interacting proteins have been hypothesized to be involved in the destabilization of the homodimeric states. In Mad1, this position corresponds to residue Asp 112. As reported for the complete gene product of Mad1, we show that wild-type b-HLH-LZ does not homodimerize or bind DNA under physiological conditions. On the other hand, the single mutation of Asp 112 to an Asn enables the b-HLH-LZ to dimerize and bind DNA. Our results suggest that Asp 112 is implicated in the destabilization of Mad1 b-HLH-LZ homodimer. Interestingly, this side chain is observed to form a salt bridge at the interface of the LZ domain in the crystal structure of Mad1/Max heterodimeric b-HLH-LZ bound to DNA [Nair, S. K., and Burley, S. K. (2003) Cell 112, 193-205]. This clearly suggests that Asp 112 plays a crucial role in the molecular recognition between Max and Mad1.
authors.affiliation: Département de Pharmacologie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
authors.forename: Martin
authors.initials: M
authors.lastname: Montagne
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Naud
authors.forename: François-Olivier
authors.initials: FO
authors.lastname: McDuff
authors.forename: Pierre
authors.initials: P
authors.lastname: Lavigne
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics; D019281:Dimerization; D024721:E-Box Elements; D006863:Hydrogen-Ion Concentration; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C067850:Myc associated factor X; D014157:Transcription Factors; D004247:DNA