Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms

Abstract

The seven 14-3-3 isoforms are highly abundant human proteins encoded by similar yet distinct genes. 14-3-3 proteins recognize phosphorylated motifs within numerous human and viral proteins. Here, we analyze by X-ray crystallography, fluorescence polarization, mutagenesis and fusicoccin-mediated modulation the structural basis and druggability of 14-3-3 binding to four E6 oncoproteins of tumorigenic human papillomaviruses. 14-3-3 isoforms bind variant and mutated phospho-motifs of E6 and unrelated protein RSK1 with different affinities, albeit following an ordered affinity ranking with conserved relative KD ratios. Remarkably, 14-3-3 isoforms obey the same hierarchy when binding to most of their established targets, as supported by literature and a recent human complexome map. This knowledge allows predicting proportions of 14-3-3 isoforms engaged with phosphoproteins in various tissues. Notwithstanding their individual functions, cellular concentrations of 14-3-3 may be collectively adjusted to buffer the strongest phosphorylation outbursts, explaining their expression variations in different tissues and tumors.

The identification and structural analysis of potential 14-3-3 interaction sites on the bone regulator protein Schnurri-3

“…1a). Moreover, the K d determination of the SHN3pS542 peptide under reducing conditions restored the affinity hierarchy, with 14-3-3 as the weakest isoform binder (Gogl et al, 2021). Isothermal titration calorimetry assays of 14-3-3 proteins with SHN3pS542.…”

“…1c). As discussed above, the nanomolar affinity of SHN3pS542 for 14-3-3 was a surprising exception compared with the 14-3-3 hierarchy of binding affinity, where 14-3-3 is normally the weakest binder to phosphorylated peptides (Gogl et al, 2021). After the observation that the SHN3pS542 peptide binds covalently to 14-3-3 in the crystal structure, we tested it in an ITC assay under reducing conditions (Fig.…”

“…Recent work by Gogl et al (2021) characterized the interaction between 14-3-3 proteins and E6 protein-derived phosphopeptides and reported how different peptides maintain a conserved affinity hierarchy towards 14-3-3 isoforms. 14-3-3 isoforms were ranked into four groups on the basis of the measured affinities (highest to lowest) towards the phosphorylated peptides studied: > > // > "/.…”

“…Despite the discovery of exceptions to the canonical binding motifs (Johnson et al, 2010), the presence of an aliphatic residue may be the cause of the weak affinity of the SHN3pT869 peptide for 14-3-3 proteins. Moreover, Gogl and coworkers described how the binding of phosphopeptides to 14-3-3 proteins is governed by subtle sequence variations that modulate the intermolecular and intramolecular contacts of 14-3-3-peptide complexes and intramolecular contacts of the peptide in its unbound state (Gogl et al, 2021). Charge distribution caused by specific residues on the unbound form of the peptides might be responsible for the formation of charge clamps which can hamper the binding to 14-3-3.…”

PPI Modulators of E6 as Potential Targeted Therapeutics for Cervical Cancer: Progress and Challenges in Targeting E6

“…A recent preprint article also investigated the potential druggability of the E6 PBM by targeting the E6–14-3-3ζ interaction. The study shows that the two proteins interact and that a small-molecule stabilizer of 14-3-3ζ, fusicoccin, weakens the E6–14-3-3ζ interaction using polarization and X-ray crystallography [ 77 ]. Figure 2 gives an overview of the inhibition of all the aforementioned PPIs of E6.…”