Semirational design of Jun-Fos coiled coils with increased affinity: Universal implications for leucine zipper prediction and design

Abstract

Activator protein-1 (AP-1) is a crucial transcription factor implicated in numerous cancers. For this reason, nine homologues of the AP-1 leucine zipper region have been characterized: Fos (c-Fos, FosB, Fra1, and Fra2), Jun (c-Jun, JunB, and JunD), and semirational library-designed winning peptides FosW and JunW. The latter two were designed to specifically target c-Fos or c-Jun. They have been identified by using protein-fragment complementation assays combined with growth competition. This assay removes nonspecific, unstable, and protease susceptible library members from the pool, leaving winners with excellent drug potential. Thermal melts of all 45 possible dimeric interactions have been surveyed, with the FosW-c-Jun complex displaying a melting temperature (T(m)) of 63 degrees C, compared to only 16 degrees C for wild-type c-Fos-c-Jun interaction. This impressive 70,000-fold K(D) decrease is largely due to optimized core packing, alpha-helical propensity, and electrostatics. Contrastingly, due to a poor c-Fos core, c-Fos-JunW dimerizes with lower affinity. However the T(m) far exceeds wild-type c-Fos-c-Jun and averaged JunW and c-Fos, indicating a preference over either homodimer. Finally, and with wider implications, we have compiled a method for predicting interaction of parallel, dimeric coiled coils, using our T(m) data as a training set, and applying it to 59 bZIP proteins previously reported. Our algorithm, unlike others to date, accounts for helix propensity, which is found to be integral in coiled coil stability. Indeed, in applying the algorithm to these 59(2) bZIP interactions, we were able to correctly identify 92% of all strong interactions and 92% of all noninteracting pairs.