A Structural Basis for 14-3-3σ Functional Specificity*♦

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, 14-3-3, 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 14-3-3-specific functions is unknown. We demonstrate that endogenous 14-3-3 preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3 bound to an optimal phosphopeptide ligand at 2.4 Å resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3 homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3-containing heterodimers, rationalizing preferential homodimerization of 14-3-3 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 14-3-3. Instead, the structure suggests a second ligand binding site involved in 14-3-3-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three -specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

“…Comparative structural analyses of 14-3-3 isoforms suggested regions, including the surface formed by helices H8 and H9 (Fig. 3C), which presumably dictate ligand and dimerization preferences (44,55,56). Our results show that the RGS domain of RGS3 interacts with the less conserved regions of helices H6 and H8 outside the central channel of the 14-3-3 dimer (Figs.…”

“…Residues that are totally conserved among all human isoforms are colored in yellow. The black ellipses in panels B and C indicate the specificity region believed to be involved in protein-protein interactions and responsible for isoform-selective contacts (44,55,56).…”

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

“…Previous studies have shown that, apart from , several of the isoforms are able to form heterodimers (2,3). The presence of unique residues at the dimer interface provided a clear structural explanation for its homodimerization preference (38). Using an in vivo-based approach, Chaudhri et al (2) found that the isoform preferentially formed heterodimers with no observable homodimer formation.…”

“…Previously, a number of three-dimensional structures of 14-3-3 isoforms have been determined and deposited in the Protein Data Bank (PDB; www.pdb.org): , in its apo form (35), as peptide complexes (14,17,18) and in complex with serotonin N-acetyltransferase (AANAT) (36); and in apo-and peptide-bound states (37,38). In addition, the structure of was published (39) but not deposited in the PDB.…”

Determinants of 14-3-3σ Protein Dimerization and Function in Drug and Radiation Resistance

“…The atomic structure of 14-3-3 has been determined (4,17) to have an overall dimeric structure that resembles a flattened horseshoe (1). Each subunit of the dimer consists of nine ␣-helices.…”