title: Two cysteines in plant R2R3 MYB domains participate in REDOX-dependent DNA binding.
abstract: Plant R2R3 MYB domain proteins comprise one of the largest known families of transcription factors. Discrete evolutionary steps have shaped the plant-specific R2R3 MYB family from the broadly distributed R1R2R3 MYB proteins. R1R2R3 MYB domains have a single Cys residue (Cys-130) that needs to be reduced for DNA binding and transcriptional activity. In contrast, most R2R3 MYB domains contain two cysteines, Cys-49 and Cys-53, with Cys-53 at the equivalent position as Cys-130 in R1R2R3 MYB. Using the maize P1 regulator of flavonoid biosynthesis as a typical R2R3 MYB-domain protein, we investigated here the in vitro REDOX requirement for DNA binding by P1. We show that the C53S mutation requires reducing conditions for DNA-binding, whereas C53A binds DNA under oxidizing and reducing conditions. Neither mutation impairs the in vivo regulatory activity of P1. The C49S and C49A mutants bind DNA in vitro irrespective of the REDOX conditions. A C49I mutant, which simulates the MYB domain of c-MYB, binds DNA only under reducing conditions, and its binding is significantly affected by the C53S replacement. It is interesting that under non-reducing conditions, Cys-49 and Cys-53 form a disulfide bond that prevents the R2R3 MYB domain from binding DNA. Together, our results suggest that the evolutionary origin of Cys-49 within the plants has provided R2R3 MYB domains with a regulatory feature not present in animal MYB domains, highlighting fundamental structural and functional differences between similar DNA-binding domains from plants and animals.
authors.affiliation: Department of Plant Cellular and Molecular Biology and Plant Biotechnology Center, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: George F
authors.initials: GF
authors.lastname: Heine
authors.forename: J Marcela
authors.initials: JM
authors.lastname: Hernandez
authors.forename: Erich
authors.initials: E
authors.lastname: Grotewold
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D003545:Cysteine / Q000502:physiology*; D018744:DNA, Plant / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C482738:BOTRYTIS SUSCEPTIBLE1 protein, Arabidopsis; D018744:DNA, Plant; D004220:Disulfides; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D014157:Transcription Factors; D003545:Cysteine