Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors

The proteins commonly referred to as 14-3-3s have recently come to prominen ce in the study of protein:protein interactions, having been shown to act a s allosteric or steric regulators and possibly scaffolds. The binding of 14 -3-3 proteins to the regulatory phosphorylation site of nitrate reductase ( NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser(543). Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner, Stimulation resulted from a reduction i n KD and an increase in steady-state binding level (R-eq), As shown previou sly for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 in teracted directly with cations, which bind and affect surface hydrophobicit y. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis fo r enhanced binding was a reduction in KD. At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detecta ble, AMP phosphatase activity.