Potential in vitro and ex vivo targeting of bZIP53 involved in stress response and seed maturation in Arabidopsis thaliana by five designed peptide inhibitors.

Basic leucine zipper (bZIP) transcription factors (TFs) are eukaryote-specific proteins that bind to DNA as a homodimer or heterodimer and regulate gene expression. They are involved in several biological processes in plants; therefore inhibiting bZIP-DNA binding activity by targeting protein-protein interface is an attractive proposition with aspects of both basic and applied biology. Here, we describe the equilibrium and kinetic interactions studies of a designed peptide inhibitor A-ZIP53 and its four variants with the bZIP53 protein, a key regulator of seed maturation phase and stress response in Arabidopsis. Five designed peptide inhibitors were primed to preferentially interact with bZIP53 and inhibit its DNA binding activity. Isothermal circular dichroism (CD) studies were used to quantify the structural changes accompanying heterodimers formation between bZIP53 and five A-ZIP53s. Equilibrium studies using electrophoretic mobility shift assay (EMSA) and fluorescence polarization (FP) assays suggest that A-ZIP53s and bZIP53 mixture form heterodimers, incapable of binding to DNA. Four A-ZIP53 derivatives were designed with additional interactions that drive heterodimerization with bZIP53. A-ZIP53s dose-dependent FP studies show that peptide inhibitors displaced the DNA bound bZIP53 with nM half-maximal inhibitory (IC50) concentrations. Using FP, time-dependent displacement kinetic studies were used to rank five A-ZIP53s for their abilities to displace DNA-bound bZIP53 with a rank order of A-ZIP53 < A-ZIP53(A → E) < A-ZIP53(N → A) < A-ZIP53(R → E) < A-ZIP53(A → E,N → A). In transient transfection assays, bZIP53-mediated GUS activity was inhibited by equimolar concentrations of five A-ZIP53s with A-ZIP53(A → E,N → A) the most effective one. Similar peptide inhibitors may be designed against other bZIP proteins to study their functions in vivo.