title: S-nitrosylation influences the structure and DNA binding activity of AtMYB30 transcription factor from Arabidopsis thaliana.
abstract: MYB proteins are a family of transcription factors that play an important role in plant development and regulatory defense processes. Arabidopsis thaliana MYB30 (AtMYB30), a member of this protein family, is involved in cell death processes during the hypersensitive response (HR) of plants. HR is characterized by a vast production of reactive oxygen species (ROS) and nitric oxide (NO). NO may thus influence the binding of AtMYB30 to DNA. In this work we evaluated the effect of NO on AtMYB30 DNA binding activity, and also in the protein structural properties. A fully active minimal DNA-binding domain (DBD) of AtMYB30 (residues 11-116) containing two cysteine residues (C49 and C53) was overexpressed and purified. Site-directed mutagenesis was used to obtain AtMYB30 DBD mutants C49A and C53A. The DNA binding activity of AtMYB30 DBD, and Cys single mutants is clearly inhibited upon incubation with a NO donor, and S-nitrosylation was confirmed by the biotin switch assay. Finally, in order to understand the mechanism of NO effect on AtMYB30 DNA binding activity we performed circular dichroism analysis, to correlate the observed protein function inhibition and a potential structural impairment on AtMYB30 DBD. Indeed, NO modification of C49 and C53 residues promotes a subtle modification on the secondary structure of this transcription factor. We thus demonstrated, using various techniques, the in vitro effect of NO on AtMYB30 DBD, and thus the potential consequences of NO activity on plant metabolism influenced by this transcription factor.
authors.affiliation: Centro de Biologia Molecular Estrutural-INBEB, Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, Brazil.
authors.forename: Carolina Pereira
authors.initials: CP
authors.lastname: Tavares
authors.affiliation: Centro de Biologia Molecular Estrutural-INBEB, Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, Brazil.
authors.forename: Javier
authors.initials: J
authors.lastname: Vernal
authors.affiliation: Centro de Biologia Molecular Estrutural-INBEB, Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, Brazil.
authors.forename: Ricardo Alexandre
authors.initials: RA
authors.lastname: Delena
authors.affiliation: Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CC1245 (7600) Mar del Plata, Argentina.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Lamattina
authors.affiliation: Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CC1245 (7600) Mar del Plata, Argentina.
authors.forename: Raul
authors.initials: R
authors.lastname: Cassia
authors.affiliation: Centro de Biologia Molecular Estrutural-INBEB, Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, Brazil. Electronic address: hterenzi@ccb.ufsc.br.
authors.forename: Hernán
authors.initials: H
authors.lastname: Terenzi
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001681:Biological Assay; D001710:Biotin / Q000737:chemistry; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018744:DNA, Plant / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D020030:Nitric Oxide Donors; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D018744:DNA, Plant; C464659:MYB30 protein, Arabidopsis; D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D014157:Transcription Factors; D009569:Nitric Oxide; D001710:Biotin; D003545:Cysteine
keywords: Arabidopsis; AtMYB30 DBD; Biotin switch assay; MYB transcription factors; Nitric oxide; Nitrosylation