PDBsum entry: 1ywt

Signaling protein/de novo protein

PDB id: 1ywt

Contents

Protein chains

223 a.a. *

Ligands

SER-HIS-SEP-TYR-PRO-ALA

MET-ALA-ARG-SER-HIS-SEP-TYR-PRO-ALA

Metals

_CA

Waters ×55

* Residue conservation analysis

PDB id:

1ywt

Name:

Signaling protein/de novo protein

Title:

Crystal structure of the human sigma isoform of 14-3-3 in complex with a mode-1 phosphopeptide

Structure:

14-3-3 protein sigma. Chain: a, b. Synonym: stratifin, epithelial cell marker protein 1. Engineered: yes. Sythetic optimal phosphopeptide (mode-1). Chain: c, d. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: sfn, hme1. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: solid phase synthesis of optimal phosphopeptide sequence as determined from library screen

Biol. unit:

Tetramer (from PQS)

Resolution:

2.40Å R-factor: 0.233 R-free: 0.283

Authors:

E.W.Wilker,R.A.Grant,S.C.Artim,M.B.Yaffe

Key ref:

E.W.Wilker et al. (2005). A structural basis for 14-3-3sigma functional specificity. J Biol Chem, 280, 18891-18898. PubMed id: 15731107 DOI: 10.1074/jbc.M500982200

Date:

18-Feb-05 Release date: 01-Mar-05

Protein chains

P31947  (1433S_HUMAN) -  14-3-3 protein sigma

Seq: 248 a.a.

Struc: 223 a.a.

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (4 terms)
• Biological process: cell proliferation (15 terms)
• Biochemical function: protein binding (4 terms)

DOI no: 10.1074/jbc.M500982200

J Biol Chem 280:18891-18898 (2005)

PubMed id: 15731107

A structural basis for 14-3-3sigma functional specificity.

E.W.Wilker, R.A.Grant, S.C.Artim, M.B.Yaffe.

ABSTRACT

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.

Selected figure(s)

Figure 3.
FIG. 3. The structure of 14-3-3 and the basis for homodimerization. A, overview of the 14-3-3 -mode-1 phosphopeptide complex. Two orthogonal views are shown with the protein in ribbons representation with one monomer colored orange and the other colored pink. The dimer 2-fold axis is indicated in each view. Side chains on either side of the interface that differ between the and isoforms are shown as yellow and green ball-and-stick representations. The phosphopeptide, shown in stick representation, is colored blue. B-E, differences between the dimer interfaces of 14-3-3 and - facilitate homodimerization of the isoform. B, a close-up bottom view of the dimer rotated 180° from the view in the right panel of A, highlighting sequence differences at the interface. C, alignment of the structures of the and isoforms reveals subtle differences in helix packing and register at the interface. The 14-3-3 ribbon is colored as described for A-C, and the two monomers of the isoform are colored cyan and blue, respectively. Sequence differences in the loop linking helices B and C(orange/blue) may be responsible for the different orientations of helix B. On the opposite side of the interface, 14-3-3 is disordered in the loop connecting helices C' and D' (pink/cyan) where there is a two-amino acid insertion relative to the sequence, and the D' helix has one less turn at its NH[2]-terminal end. D, at the 14-3-3 dimer interface a substitution of Phe ( ) for Cys ( ) at residue 25 produces a ring-ring interaction between Phe-25 ( B) and Tyr-84 ( D'). The aromatic ring of Phe-25 prevents the alkyl chain of Lys-9 ( A) from participating in van der Waals interactions with Tyr-84 as seen at the interface. Instead, Lys-9 makes a salt bridge across the interface with the side chain of Glu-83 at the end of the ordered part of helix D'. E, in a modeled structure of the 14-3-3 / heterodimer interface, Glu-80 in 14-3-3 D' (pink) would be juxtaposed against Glu-5 in A of 14-3-3 (blue). This destabilizing interaction would similarly disrupt heterodimerization of with the , , and isoforms as well.

Figure 4.
FIG. 4. Ligand recognition by 14-3-3 . A, the phosphopeptide-binding pocket. Stereo view of the mode-1 phosphopeptide bound to one monomeric subunit of the 14-3-3 dimer. The phosphopeptide is shown in stick representation with carbon atoms colored yellow, nitrogens blue, oxygens red, and phosphorus purple. 14-3-3 is shown in surface representation shaded by similar atom-type color coding except with carbon colored green. B, molecular basis for phosphospecificity. The phosphate group of the phosphoserine is coordinated by four conserved 14-3-3 side chains. The protein backbone is represented by green ribbons; the phosphopeptide and the side chains that interact with the phosphate group are shown in stick representation. Key interactions observed between the phosphopeptide and 14-3-3 are the same as those in the crystal structure of the 14-3-3 -phosphopeptide complex (12). C, a novel binding surface unique to 14-3-3 . Surface representations of the 14-3-3 dimer are shown along with a ball-and-stick representation of the phosphopeptide (green) in the same orientations as in Fig. 3A. The surfaces are color coded by sequence conservation with highly conserved residues shaded magenta and non-conserved residues yellow. Three exposed residues of 14-3-3 (Met-202, Asp-204, and His-206), which differ from the absolutely conserved sequence in all other known isoforms, are shaded blue. These highly exposed residues protrude from the top of a prominent ridge above the concave face that contains the phosphopeptide-binding grooves. D, mutation of the novel binding surface of 14-3-3 allows CDC25C ligand binding. U2OS cells were transfected with HA-14-3-3 wild-type (wt) or a 14-3-3 Met-202 Ile,Asp-204 Glu,His-206 Asp triple mutant (mut3). 24 h following transfection, cells were irradiated (IR) with 10 grays of ionizing radiation or received no treatment, and were lysed 4 h later. Whole cell lysates were immunoprecipitated (IP) with an anti-HA antibody, separated by SDS-PAGE, and blotted with antibodies against Cdc25C (upper panel), c-Raf-1 (middle panel), and the HA epitope (lower panel).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 18891-18898) copyright 2005.

Figures were selected by an automated process.