Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif

Abstract

The second phase is independent of concentration and is exponential. In contrast, in the unfolding direction, all molecules display two-state kinetics. Collectively this implies a transition state between unfolded helices and dimeric intermediate that is readily traversed in both directions. We demonstrate that the added stability of cFos-JunW Ph1 relative to cFos-JunW is achieved via a combination of kinetic rate changes; cFosJunW E23K has an increased initial dimerization rate, prior to the major transition state barrier while cFos-JunW Q21R displays a decreased unfolding rate. The former implies that improved hydrophobic burial and helix-stabilizing mutations exert their effect on the initial, rapid, monomer-collision event. In contrast, electrostatic interactions exert their effect late in the folding pathway. Although our focus is the leucine zipper region of the oncogenic transcription factor Activator Protein-1, coiled coils are ubiquitous and highly specific in their recognition of partners. Consequently, generating kinetic-based rules to predict and engineer their stability is of major significance in peptidebased drug design and nano-biotechnology.

Selectional and Mutational Scope of Peptides Sequestering the Jun–Fos Coiled-Coil Domain

“…44 Recently, we investigated the kinetics of complex formation between cFos and JunW, JunW Ph1 , and the intermediate mutants (JunW Q21R , JunW E23K ) using stopped-flow analysis. 45 This study revealed that all of the possible peptide mixtures fold via a transiently populated dimeric intermediate. Of these, cFos-JunW E23K is the only dimer where the intermediate can be detected by equilibrium denaturation.…”

“…1). 45 The probability of these two contrasting selection systems arriving at such similar sequences by chance is unfeasibly low, indicating that both systems indeed selected for similar properties. The sequence of helix JunW Ph2 differs from the sequences of JunW Ph1 and JunW especially in the hydrophobic core.…”

iPEP: peptides designed and selected for interfering with protein interaction and function

“…A further insight into the structural determinants of stability arose by dissecting the folding pathway of four c-Jun leucine zipper variants that bind with high affinity to c-Fos [24]. These encompassed a PCA-selected winner (JunW) [17], a phage-display-selected winner (JunW Ph1 ) [22] and two intermediate mutants.…”

Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex

“…Echoing an early suggestion that Fos and Jun leucine zipper domains be used as a molecular Velcro in biotechnology (53) are studies that utilized leucine zippers to verify the substrate for a tyrosine kinase (54), to facilitate the amyloid formation of ␣-synuclein (55), to enhance the interaction of different proteins in a yeast two-hybrid setup (56), and to help design synthetic leucine zipper peptides that exhibit extremely high affinity for heterodimerization (41,(57)(58)(59). This work presents a suite that incorporated Fos and Jun to solve different challenges faced in recombinant protein expression and purification.…”