Design of a leucine zipper coiled coil stabilized 1.4 kcal mol −1 by phosphorylation of a serine in the e position

Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (δδ G P = ‐1.4 kcal mol −1 dimer −1 or ‐0.7 kcal mol −1 residue −1 ). Mutagenesis studies indicate that three arginines form a network of inter‐helical ( i, i ' + 5; i, i ' + 2) and intra‐helical ( i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (δδ G P = ‐0.5 kcal mol −1 dimer −1 ). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15‐fold higher affinity. Using a transient transfection assay, we document a PKA dependent four‐fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by δδ G P = + 1.2 kcal mol −1 dimer −1 . We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.