The phytotoxin fusicoccin differently regulates 14-3-3 proteins association to mode III targets

Abstract

Modulation of the interaction of regulatory 14-3-3 proteins to their physiological partners through small cell-permeant molecules is a promising strategy to control cellular processes where 14-3-3s are engaged. Here, we show that the fungal phytotoxin fusicoccin (FC), known to stabilize 14-3-3 association to the plant plasma membrane H 1 -ATPase, is able to stabilize 14-3-3 interaction to several client proteins with a mode III binding motif. Isothermal titration calorimetry analysis of the interaction between 14-3-3s and different peptides reproducing a mode III binding site demonstrated the FC ability to stimulate 14-3-3 the association. Moreover, molecular docking studies provided the structural rationale for the differential FC effect, which exclusively depends on the biochemical properties of the residue in peptide C-terminal position. Our study proposes FC as a promising tool to control cellular processes regulated by 14-3-3 proteins, opening new perspectives on its potential pharmacological applications. V C 2014 IUBMB Life, 66(1): [52][53][54][55][56][57][58][59][60][61][62] 2014

Fusicoccin Activates KAT1 Channels by Stabilizing their Interaction with 14-3-3- Proteins

“…This confirms that the peptide, when not phosphorylated, does not bind to the proteins, as predicted by the previous experiment with the KAT1 mutant S676A (see Figure 1). Figures 3F and 3G show results obtained when the experiment was repeated with the phosphorylated peptide, CPP, in the absence ( Figure 3F) and presence ( Figure 3G Notably, the stabilizing effect of FC on CCP binding to 14-3-3c (2.5-fold) is lower than that reported for the H + pump phosphopeptide binding (>10-fold) (Würtele et al, 2003;Paiardini et al, 2014), suggesting that FC may bind the CPP of KAT1 in an alternative manner, likely due to the presence of the polar asparagine in the last position that replaces the more commonly found valine.…”

“…It is generally believed that FC stabilizes the complex between 14-3-3 proteins and MODE III motifs that are found at the end of a protein sequence (Paiardini et al, 2014). In all crystal structures obtained so far, FC occupies the end of the 14-3-3 groove, suggesting that any downstream extension of the bound phospho-peptide would cause a steric clash with the toxin, specifically with the hydroxyl group on C12.…”

“…The phosphoserine of CPP establishes two electrostatic interactions with R63 and R136 as well as a hydrogen bond with Y137 of 14-3-3c ( Figure 2B). These residues are highly conserved in 14-3-3 proteins as they bind to the phosphate moiety of the target peptides (Paiardini et al, 2014). Additionally, CPP interacts with several polar and hydrophobic residues of the 14-3-3c binding groove ( Figure 2B).…”

“…In both cases, the MODE III phosphopeptide of the 14-3-3 target protein ends with the small hydrophobic amino acid valine that establishes crucial interactions with the hydrophobic rings of FC (Würtele et al, 2003;Anders et al, 2013). The presence of a C-terminal small hydrophobic residue is considered necessary for FC binding (Paiardini et al, 2014). Important to note in this context is that the MODE III binding motif of KAT1 does not end with such a canonical hydrophobic residue, but with the hydrophilic asparagine.…”

“…In the case of the plant H + -ATPase, the interaction of 14-3-3 with the cytosolic C terminus of the H + pump is known in atomic detail (Ottmann et al, 2007), as is the molecular mechanism mediating upregulation of the H + -ATPase by the fungal toxin fusicoccin (FC), the best known activator of the H + pump and a potent enhancer of stomata opening (Aducci et al, 1995). FC is a small amphipatic molecule that binds to the protein complex formed by the H + pump C terminus and 14-3-3 proteins and strongly stabilizes the interaction of the two partners (Würtele et al, 2003;Paiardini et al, 2014). Currently it is not known how the activity of the KAT1 channel is integrated in this scenario.…”