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

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Abstract
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 (δδGP = ‐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 (δδGP = ‐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 δδGP = + 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.