AN EFFICIENT SCREENING ASSAY FOR THE RAPID AND PRECISE DETERMINATION OF AFFINITIES BETWEEN LEUCINE-ZIPPER DOMAINS

The protein products of the jun and fos oncogenes require a functional protein-protein interaction domain, called the ''leucine zipper domai n'', to exert their transcriptional regulatory activity. A scintillati on proximity assay was developed in which the biotinylated leucine zip per domain of the Jun protein (275-315) was immobilized on streptavidi n-coated microfluorospheres and in which the leucine zipper domain of the Fos protein (160-200) was used as free, labeled ligand. The Fos le ucine zipper peptide specifically bound to the Jun leucine zipper pept ide, and for the first time, a dissociation constant (K(d) = 110 +/- 1 2 nM in PBS/0.1% Tween) could be determined. Optimal heterodimer forma tion was reached at neutral pH. Both acidic and alkaline pH decreased the association of the peptides which was, furthermore, completely abo lished by 500 mM NaCl, confirming that charged residues are critical f or heterodimerization. A commercially obtained recombinant Jun protein competed as efficiently as the Jun leucine zipper peptide for binding to the Fos peptide, confirming the feasibility of using the two leuci ne zipper peptides to study the interactions between the two transcrip tion factors. We also injected leucine zipper peptides individually in to Xenopus oocytes to study whether they would interfere with the acti vity of the Fos/Jun heterodimer in vivo. Both peptides blocked selecti vely insulin-mediated oocyte maturation with an IC50 in the range of 1 5 ng per oocyte. In conclusion, the scintillation proximity assay desc ribed here may be used to investigate protein-protein interactions med iated by leucine zipper structures and to identify compounds that inhi bit leucine zipper association.