Structural and interaction analysis of monomeric 14-3-3ζ phosphorylated at Ser58 by NMR spectroscopy.

14-3-3 proteins are important dimeric regulatory proteins in the human body that bind phosphorylated partners and regulate their activity. Phosphorylation of 14-3-3 proteins at S58 (pS58) at the dimeric interface was shown to trigger their monomerization and change their thermal stability, hydrophobicity, binding affinity and stoichiometry. However, the direct effect of phosphorylation and monomerization on 14-3-3 structure remains poorly understood. Here, we employed solution nuclear magnetic resonance (NMR) spectroscopy to elucidate the structural features of monomeric 14-3-3ζ pS58 protein and its interaction with (phospho)Tau protein variants with single-residue resolution. First, based on the NMR assignment and calculated secondary structure propensities, we revealed that the phosphorylation and monomerization decrease helical propensity of the αC and αD helices. Second, using NMR titration experiments, we identified two secondary interaction sites (SISs) on 14-3-3ζ protein outside of the binding groove - one located at the end of αC helix and another in the loop connecting the αH and αI helices. Third, paramagnetic relaxation enhancement experiments showed which 14-3-3ζ regions are approached by its flexible C-terminus. Importantly, these regions overlap with the SISs but not the binding groove, providing an alternative explanation for the autoinhibitory function of the C-terminus. Taken together, we report novel insight into the structure and interactions of monomeric 14-3-3, connected with S58 phosphorylation.