RESUMO
Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.
Assuntos
Metilação de DNA , DNA/química , Proteínas de Homeodomínio/química , Radical Hidroxila/química , Zíper de Leucina/genética , Sequência de Bases , Sítios de Ligação/genética , DNA/metabolismo , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Helianthus/química , Helianthus/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Modelos Moleculares , Estrutura Molecular , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Plant homeodomain-leucine zipper (HD-Zip) proteins, unlike many animal homeodomains (HDs), are unable to bind DNA as monomers. To investigate the molecular basis of their different behavior, we have constructed chimeras between the HD of the sunflower HD-Zip protein Hahb-4 and that of Drosophila engrailed (EN). Analysis of the interaction of these proteins with the pseudopalindromic Hahb-4 binding site and the monomeric EN binding site suggests that the loop located between helix I and helix II (amino acids 21-28) of EN is enough to confer efficient DNA binding activity to the Hahb-4 HD. Accordingly, the combined mutation of residues 24 and 25 of Hahb-4 to those present in EN (S24R/R25Y) originated an HD able to interact with the EN binding site, while single mutations were ineffective. We have also determined that a protein with the leucine zipper and helix III of Hahb-4 fused to the rest of the EN HD binds to the Hahb-4 pseudopalindomic binding site with increased affinity and shows extended contacts with DNA respective to Hahb-4. We conclude that the loop located between helix I and helix II of the HD must be regarded as one of the segments that contribute to the present-day diversity in the properties of different HDs.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , DNA/metabolismo , Dimerização , Proteínas de Drosophila , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Homeodomínio/química , Zíper de Leucina , Dados de Sequência Molecular , Proteínas de Plantas/química , Mutação Puntual/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/químicaRESUMO
Several families of plant transcription factors contain a conserved DNA binding motif known as the homeodomain. In two of these families, named Hd-Zip and glabra2, the homeodomain is associated with a leucine zipper-like dimerization motif. A group of Hd-Zip proteins, namely Hd-ZipII, contain a set of conserved cysteines within the dimerization motif and adjacent to it. Incubation of one of these proteins, Hahb-10, in the presence of thiol-reducing agents such as dithiothreitol or reduced glutathione produced a significant increase in DNA binding. Under such conditions, the protein migrated as a monomer in non-reducing SDS-polyacrylamide gels. Under oxidizing conditions, a significant proportion of the protein migrated as dimers, suggesting the formation of intermolecular disulfide bonds. A similar behavior was observed for the glabra2 protein HAHR1, which also contains two conserved cysteines within its dimerization domain. Site-directed mutagenesis of the cysteines to serines indicated that each of them has different roles in the activation of the proteins. Purified thioredoxin was able to direct the NADPH-dependent activation of Hahb-10 and HAHR1 in the presence of thioredoxin reductase. The results suggest that redox conditions may operate to regulate the activity of these groups of plant transcription factors within plant cells.