Structural basis for protein-protein interactions in the 14-3-3 protein family

Abstract

The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein-protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.

Fig. 1.

Overview of the dimeric 14-3-3 structure. Helices and loops involved in target domain interactions are labeled. Each monomer is colored blue to red from the N to C terminus. An aperture exists at the central dimeric interface, which is marked with a circle.

Fig. 2.

Schematic representation of the heterodimerization process involving the ε (green) and ζ (yellow) isoforms. The lines between identified residues indicate specific interactions.

Fig. 3.

The selective nature of the primary interaction site. (A) Close-up view with side chain interactions highlighted for Pep1 in the binding groove of ε. The N- to C-terminal orientation is the same in all other 14-3-3 structures with phosphopeptides. (B) Same view as in A except with the ε peptide binding groove surface colored yellow. Reverse orientation of the same peptide (blue wire frame) about the phosphoserine Cα–Cβ bond results in major clashes. (C) Same view as in A, now with the character of the residues that make up the peptide binding groove color coded onto the surface as yellow (hydrophobic), red (negatively charged), blue (positively charged), and gray (neutral).

Fig. 4.

Calculated desolvation energies mapped onto the monomeric surface representations for each of the seven human isoforms. All views look down onto the dimerization interface (Left), central binding groove, and the αG–αI helices (Right). The desolvation energies are color coded from high (blue) to low (red). The sequence conservation is mapped onto a tube representation of the τ isoform, color coded from green (100%) to white. The variable CD and HI loops are labeled V1 and V2, respectively, and the two conserved low-energy desolvation sites are labeled S1 (S1a and S1b) and S2, respectively.

Fig. 5.

Dynamic nature of the 14-3-3 dimers. (A) Crystal structure of the apo-β isoform looking down the peptide binding grooves, which are labeled open and closed for the individual monomers. (B) Superimposition of all seven closed state 14-3-3 isoforms using only one monomer as the reference, with β shown in blue and τ in green. The other 14-3-3 monomers, which have intermediate positions, are colored transparent gray.