title: Exposed loop domains of complexed 14-3-3 proteins contribute to structural diversity and functional specificity.
abstract: The 14-3-3 family of proteins functions through protein:phosphoprotein interactions, the nature of which has been elucidated using x-ray crystallography. However, some key structural features in nonconserved regions have yet to be fully resolved, leaving open questions regarding the functional selectivity of 14-3-3 family members for diverse clients. In an effort to study surface accessible structural features in 14-3-3 containing macromolecular complexes and to illuminate important structure/function variations among the 14-3-3 isoforms, we determined the epitopes for three unique monoclonal antibodies (mAbs) developed against the Arabidopsis (Arabidopsis thaliana) G-box DNA:protein complex. The epitopes mapped to different loops in a phylogenetically important subset of the 13 14-3-3 family members. All three epitopes were on a common exposed face of complexed 14-3-3s. Two of the mAbs recognized linear sequences within loops 5 and 6, while the third mAb recognized 14-3-3 residues surrounding the pivotal medial Gly in the divalent cation-binding domain of loop 8, together with distal residue(s) in the putative dynamic 10th helix that has yet to be determined by crystallography. Gly at this loop 8 position is unique to nonepsilon 14-3-3 isoforms of the plant kingdom, suggesting that this region constitutes a plant-specific key functional 14-3-3 feature and highlighting that the loop 8 region is functionally significant. Mutagenesis of the medial amino acid in the loop 8 domain changed the flexibility of the C terminus and altered client peptide-binding selectivity, demonstrating the functional significance of the surface accessible, evolutionarily distinct loop 8 domain.
authors.affiliation: Program in Plant Molecular and Cellular Biology, Department of Horticultural Science , University of Florida, Gainesville, Florida 32611, USA.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Sehnke
authors.forename: Beth
authors.initials: B
authors.lastname: Laughner
authors.forename: Helene
authors.initials: H
authors.lastname: Cardasis
authors.forename: David
authors.initials: D
authors.lastname: Powell
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Ferl
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D018604:Epitope Mapping; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D010748:Phosphopeptides / Q000378:metabolism; D010802:Phylogeny; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D048948:14-3-3 Proteins; D000911:Antibodies, Monoclonal; D029681:Arabidopsis Proteins; D019151:Peptide Library; D010748:Phosphopeptides; D020033:Protein Isoforms