title: The bZIP region of the plant transcription factor opaque-2 forms stable homodimers in solution and retains its helical structure upon subunit dissociation.
abstract: Opaque-2 (O2) is a plant bZIP transcription factor that regulates the expression of alpha and beta prolamines, the main storage proteins in seeds of cereals such as maize and Coix. One of the main processes modulating O2 activity is the heterodimerization with other bZIP transcription factors, but the primary mechanism underlying the partner choice is still unknown. In this paper, we have characterized the bZIP domain of O2 by nuclear magnetic resonance (NMR), circular dichroism (CD), and size-exclusion chromatography. Results obtained from CD measurements suggested that the native O2bZIP has about 40 of its 49 leucine-zipper residues in helical structure, while the DNA-binding domain is completely unstructured. Diffusion-ordered NMR spectroscopy and size-exclusion chromatography showed that O2 forms homodimers in solution. Thermal denaturation experiments indicate that O2 reversibly undergoes dissociation and unfolding in a process that is fully dependent on the protein concentration. Subunit dissociation of O2bZIP dimers, upon dilution of the protein, led to partially folded monomers that retained approximately 80% of the native CD ellipticity at 222 nm. We believe that the existence of partially folded monomers could decrease the entropic penalty for helix formation involved in the DNA binding and in the subunit association of O2bZIP. Stabilization of partially folded monomers may also play a significant role in the dimerization of O2 with other bZIP transcription factors and, consequently, can be important for the regulation of the biological functions of O2 in plants.
authors.affiliation: Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Departamento Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. vitorhm@bioqmed.ufrj.br
authors.forename: Vitor Hugo
authors.initials: VH
authors.lastname: Moreau
authors.forename: Alba C
authors.initials: AC
authors.lastname: da Silva
authors.forename: Rodrigo M P
authors.initials: RM
authors.lastname: Siloto
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Valente
authors.forename: Adilson
authors.initials: A
authors.lastname: Leite
authors.forename: Fábio C L
authors.initials: FC
authors.lastname: Almeida
mesh_terms: D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D031742:Coix / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D051790:G-Box Binding Factors; D007526:Isoelectric Point; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D009687:Nuclear Proteins; C102290:OHP1 protein, Zea mays; D010940:Plant Proteins; D021122:Protein Subunits; D012996:Solutions; D014157:Transcription Factors; C067402:opaque-2 protein, Zea mays