Article

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

Structural Genomics Consortium, University of Oxford, Botnar Research Centre, Oxford OX3 7LD, United Kingdom.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2006; 103(46):17237-42. DOI: 10.1073/pnas.0605779103
Source: PubMed

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.

Full-text

Available from: Jörg Günter Grossmann, May 30, 2015
0 Followers
 · 
118 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross linking and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.
    Journal of Proteome Research 03/2015; 14(4). DOI:10.1021/pr5010136 · 5.00 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 14-3-3s 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 14-3-3s 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 14-3-3s 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 14-3-3s provide new clues for better understanding of the molecular mechanisms of dimerization and multifunctionality of these universal adapter proteins. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 03/2015; 1854(5). DOI:10.1016/j.bbapap.2015.02.017 · 3.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human tyrosine hydroxylase activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator 14-3-3 proteins. We investigated the binding of singly or doubly phosphorylated and thiophosphorylated peptides, comprising the first 50 amino acids of human tyrosine hydroxylase, isoform 1 (hTH1), that contain the critical interaction domain, to 14-3-3?, by (31)P NMR. Single phosphorylation at S19 generates a high affinity 14-3-3? binding epitope, whereas singly S40-phosphorylated peptide interacts with 14-3-3? one order-of-magnitude weaker than the S19-phosphorylated peptide. Analysis of the binding data revealed that the 14-3-3? dimer and the S19- and S40-doubly phosphorylated peptide interact in multiple ways, with three major complexes formed: 1), a single peptide bound to a 14-3-3? dimer via the S19 phosphate with the S40 phosphate occupying the other binding site; 2), a single peptide bound to a 14-3-3? dimer via the S19 phosphorous with the S40 free in solution; or 3), a 14-3-3? dimer with two peptides bound via the S19 phosphorous to each binding site. Our system and data provide information as to the possible mechanisms by which 14-3-3 can engage binding partners that possess two phosphorylation sites on flexible tails. Whether these will be realized in any particular interacting pair will naturally depend on the details of each system.