Hidden disorder propensity of the N-terminal segment of universal adapter protein 14-3-3 is manifested in its monomeric form: Novel insights into protein dimerization and multifunctionality

The multiplicity of functions of 14-3-3 proteins, integrated into many cellular interactions and signaling networks, is primarily based upon their dimeric α-helical structure that is capable of binding phosphorylated protein partners as well as displaying a “moonlighting” chaperone-like activity. The structure and functions of 14-3-3 proteins are regulated in different ways, including Ser58 phosphorylation in the interface, which shifts equilibrium towards the formation of protein monomers whose role is poorly understood. While modification of Ser58 induced only partial dissociation, the engineered triple mutation of human 14-3-3ζ located in the first α-helix deeply monomerized the protein, allowing for a structural analysis of the monomeric form. Dimer-incapable 14-3-3 proteins retained binding capacity and specificity towards some phosphopartners, and also demonstrated increased chaperone-like activity on various substrates. Here, we found a substantial propensity of the N-terminal segment (~ 40 residues) of 14-3-3 proteins to intrinsic disorder, showing remarkable conservation across different isoforms and organisms. We hypothesized that this intrinsic disorder propensity, hidden in the α-helical 14-3-3 dimer, can be manifested upon its dissociation and interrogated novel monomeric 14-3-3ζ carrying both monomerizing and S58E mutations (14-3-3ζmS58E). CD spectroscopy showed that, at physiological temperatures, this protein has ~ 10–15% reduced helicity relative to the wild type protein, corresponding to roughly 40 residues. Along with the known flexibility of C-terminus, SAXS-based modeling of the 14-3-3ζmS58E structure strongly suggested pliability of its N-terminus. The unraveled disorder propensity of the N-terminal tails of 14-3-3 proteins provides new clues for better understanding of the molecular mechanisms of dimerization and multifunctionality of these universal adapter proteins.