Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of α-type plant phospholipase D.

Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca binding sites. In vitro, the predominantly expressed α-type PLDs need 20-100 mM CaCl for optimum activity, whereas the essential activator of β- or γ-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP), plays a secondary role. In the present paper, we have studied the interplay between PIP and metal ion activation of the well-known α-type PLD from cabbage (PLDα). With mixed micelles containing phosphatidyl-p-nitrophenol as substrate, PIP-concentrations in the nanomolar range are able to activate the enzyme in addition to the essential Ca activation. Mg ions are able to replace Ca ions but they do not activate PLDα. Rather, they abolish the activation of the enzyme by Ca ions in the absence, but not in the presence, of PIP. The presence of PIP causes a shift in the pH optimum of PLDα activity to the acidic range. Employing fluorescence measurements and replacing Ca by Tb ions, confirmed the presence of two metal ion-binding sites, in which the one of lower affinity proved crucial for PLD activation. Moreover, we have generated a homology model of the C2 domain of this enzyme, which was used for Molecular Dynamics (MD) simulations and docking studies. As is common for C2 domains, it shows two antiparallel β-sheets consisting of four β-strands each and loop regions that harbor two Ca binding sites. Based on the findings of the MD simulation, one of the bound Ca ions is coordinated by five amino acid residues. The second Ca ion induces a loop movement upon its binding to three amino acid residues. Docking studies with PIP reveal, in addition to the previously postulated PIP-binding site in the middle of the β-sheet structure, another PIP-binding site near the two Ca ions, which is in accordance with the experimental interplay of PIP, Ca and Mg ions.