title: A monomer-dimer equilibrium modulates the interaction of the sunflower homeodomain leucine-zipper protein Hahb-4 with DNA.
abstract: We have analysed the interaction of the sunflower homeodomain leucine-zipper (Hd-Zip) protein Hahb-4 with DNA. The complete Hd-Zip domain from Hahb-4 was able to select specific sequences from a random oligonucleotide mixture that contained a 9-bp core with four fixed and five degenerate positions. Analysis of the binding of some of the selected sequences suggests that Hahb-4 preferentially binds the dyad-symmetrical sequence CAAT(A/T)ATTG. Single-nucleotide replacements at positions 1, 5 or 9 of this sequence produced a decrease in binding of 2-4-fold. DNA binding as a function of protein concentration was non-hyperbolic. This behaviour could be explained by an equation in which dimer formation is a pre-requisite for DNA binding. A global dissociation constant (Kd) of 1.31x10(-14) M2 could be calculated. The removal of the leucine zipper promoted a change in specificity and a decrease in binding affinity (Kd=5. 03x10(-5) M). Mutation of Phe-20 of the homeodomain into Leu completely abolished DNA binding. The mutant protein, however, was able to inhibit DNA binding by the non-mutant form, presumably through the formation of heterodimers. The analysis of this inhibitory effect at different mutant concentrations allowed the estimation of the Kd for the dimer-monomer equilibrium [about (2-4)x10(-6) M]; from this, a Kd of 3-6x10(-9) M for the dimer-DNA complex could be estimated. The results obtained indicate that the formation of dimers is the main factor influencing the interaction of Hahb-4 with DNA. It is proposed that shifts in a dimer-monomer equilibrium could be used within the cell to modulate the interaction of this protein with target genes.
authors.affiliation: Area Biología Molecular, Facultad de Ciencias Bioqu ímicas y Farmacéuticas (UNR) and Programa Multidisciplinario de Biolog ía Experimental (PROMUBIE, CONICET), Suipacha 531, 2000 Rosari.
authors.forename: C M
authors.initials: CM
authors.lastname: Palena
authors.forename: D H
authors.initials: DH
authors.lastname: Gonzalez
authors.forename: R L
authors.initials: RL
authors.lastname: Chan
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D006368:Helianthus; D018398:Homeodomain Proteins / Q000378:metabolism*; D016350:Leucine Zippers; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: C091850:HD-Zip protein, Helianthus annuus; D018398:Homeodomain Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA