A monomer-dimer equilibrium modulates the interaction of the sunflower homeodomain leucine-zipper protein Hahb-4 with DNA

PALENA, CLAUDIA M.; GONZALEZ, DANIEL H.; CHAN, RAQUEL L.

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Año: 1999 vol. 341 p. 81 - 87

0264-6021

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 (K(d)) of 1.31 x 10-14 M2 could be calculated. The removal of the leucine zipper promoted a change in specificity and a decrease in binding affinity (K(d) = 5.03 x 10-5M). 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 K(d) for the dimer-monomer equilibrium [about (2-4) x 10-6 M]; from this, a K(d) of 3-6 x 10-9 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.