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 K_D 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.

Fig. 1. E6 PBMs reveal parallel binding profiles to human 14-3-3 isoforms.

a Exemplary phosphorylatable C-terminal E6 PBMs from high-risk mucosal HPV types contain the 14-3-3-binding motif III. The domains of the E6 protein are shown by green (E6N) and beige (E6C) colors, the C-terminal tail containing the phosphorylatable residue (red circle) is cyan. The positions are numbered above the sequences, according to conventional PBM numbering, with the phosphorylatable antepenultimate residue (position –2) indicated by red. b Affinities of four selected HPV E6 phospho-PBMs, p35E6 mutant variants, and RSK1 phosphopeptides toward the seven human 14-3-3 isoforms as determined by fluorescence polarization using FITC-labeled HSPB6 phosphopeptide as a tracer. Apparent K_D values determined from competitive FP experiments are presented. The heatmap representation of the data on b shows the affinity trends in the interaction profiles between 14-3-3 isoforms and four HPV E6 phospho-PBMs from strongest (red) to weakest (white). Protein names are boldfaced for clarity. c Averaged ΔΔG values between 14-3-3 isoforms and E6 phospho-PBM pairs, calculated based on their observed order of binding affinities (from weakest to strongest). Individual K_D values from Supplementary Fig. 2 were first converted into ΔG values (at T = 295 K; excluding cases when K_D > 300 μM), then average ΔΔG values (ΔΔG_av) were calculated between the indicated motifs/isoforms. Standard deviation (std) values are indicated at each number on b and c (each time, three independent measurements). All binding data are provided as Supplementary Data File 1.

Fig. 2. Structural basis for the 14-3-3ζ/phospho-18E6 PBM interaction.

a An overall view on the 14-3-3ζ dimer (subunits are in tints of gray) with two bound 18E6 phosphopeptides (cyan sticks, shown with the 2Fo-Fc electron density maps contoured at 1σ). b An overlay of the two 14-3-3 bound phosphopeptides from 16E6 (PDB ID: 6TWZ; magenta sticks) and 18E6 (this work; cyan sticks) showing the similarity of the conformation. # denotes the C-terminus (-COOH). w—water molecule, π—π-stacking interaction. Key positions are numbered according to the PBM convention. c Averaged ΔΔG values between 14-3-3 isoforms and 35E6 phospho-PBM pairs, calculated based on their observed order of binding affinities (from weakest to strongest). Individual K_D values from Supplementary Fig. 2 were first converted into ΔG values (at T = 295 K; excluding cases when K_D > 300 μM), then average ΔΔG values (ΔΔG_av) were calculated between the indicated HPV35E6 motifs.

Fig. 3. The 14-3-3ζ/18E6 PBM interaction is druggable by FSC.

a Affinities of four selected HPV E6 phospho-PBMs toward human 14-3-3ζ and 14-3-3γ in the absence and presence of FSC as determined by FP using FITC-labeled HSPB6 phosphopeptide as a tracer. Protein names are boldfaced for clarity. Apparent K_D values determined from competitive FP experiments are presented. Standard deviation (std) values are indicated. The binding curves are shown in Supplementary Fig. 2. b Averaged ΔΔG values between 14-3-3–E6 phospho-PBM pairs in the absence or presence of FSC, calculated based on their observed order of binding affinities (from weakest to strongest). Individual K_D values from Supplementary Fig. 2 were first converted into ΔG values (at T = 295 K; excluding cases when K_D > 300 μM), then average ΔΔG values (ΔΔG_av) were calculated for the E6-binding affinity changes of the indicated 14-3-3 isoforms in the absence or presence of FSC. c An overall view on the ternary complex between 14-3-3ζ (subunits are shown by surface using two tints of gray), 18E6 phosphopeptide (cyan sticks), and FSC (pink sticks). FSC was soaked into the 14-3-3ζ–18E6 chimera crystals. 2F_o-F_c electron density maps contoured at 1σ are shown for the phosphopeptide and FSC only. d The effect of FSC binding. Conformational changes in the 9th α-helix of 14-3-3 and in the C-terminal part of the 18E6 phosphopeptide upon FSC binding are shown by red arrows, a significant rise of the local B-factors of the phosphopeptide is shown using a gradient from blue to red as indicated. The amplitudes of the conformational changes are indicated in Å by dashed arrows.

Fig. 4. Hierarchized target binding by 14-3-3 isoforms is a general trend.

a Affinity maps of 14-3-3 interactions based on experimentally determined dissociation constants against the 14-3-3ome, as obtained in this study (white background) and in others^,– (gray background). The color scale is either based on affinity values or on K_D ratios. √ denotes affinities weaker than the limit of quantitation of the experimental assays. b Same map as in a, normalized to the strongest 14-3-3-binding motif. An average 34,000-fold K_D ratio is observed between the strongest and weakest 14-3-3-binding peptide. c Same map as in a, normalized to the strongest phosphopeptide-binding 14-3-3 isoform. Note that all peptides follow very similar affinity trends between the different 14-3-3 isoforms, with an average 12-fold K_D ratio between the strongest and weakest-binding 14-3-3 isoform. d Number of unique partners detected according to the BioPlex database for each 14-3-3 isoform, taken individually (left) or grouped in three subsets (right) following their relative affinity trends (strong, intermediate, and weak binders). e Number of 14-3-3 partners in BioPlex, which bound to 1, 2, 3, 4, 5, 6, or all 7 isoforms, respectively. Within each bar, the proportion of partners that bound to each individual isoform is indicated (same isoform color code as in d). f Venn diagram showing repartition of the 14-3-3 partners from BioPlex among the strong, medium, and weak phosphopeptide-binding subsets, defined as in d. g Correlation between the number of binders of 14-3-3 isoforms, according to BioPlex, and ΔΔG_av between the strongest phosphopeptide-binder, 14-3-3γ, and all individual isoforms (same color code as in d). ΔΔG values were calculated from the average K_D ratios from c. h The average amounts of prey proteins from BioPlex (normalized to the amount captured using 14-3-3γ) that interact with at least five different 14-3-3 baits, deduced from their PSM values, also show a correlation with the ΔΔG_av values of the same proteins. i Correlation of the sequence identity of human 14-3-3 isoforms relative to 14-3-3γ with the ΔΔG_av values from g. Source data are provided as a Source Data file.

Fig. 5. Cellular 14-3-3/phosphotarget complexomes.

a Abundance of the seven 14-3-3 isoforms across different human tissues and in the whole human organism, according to the PAXdb database (https://pax-db.org and ). Colors correspond to the protein abundances, according to the scale provided on the right. b Predicted proportions of 14-3-3-bound phosphoproteins that would be engaged with each individual isoform in different tissues, assuming that the majority of 14-3-3 molecules are available for interaction (same color code as in Fig. 4d). Source data are provided as a Source Data file.