科研之友
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摘要
Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca2+ binding sites. In vitro, the predominantly expressed alpha-type PLDs need 20-100 mM CaCl2 for optimum activity, whereas the essential activator of beta- or gamma-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP2), plays a secondary role. In the present paper, we have studied the interplay between PIP2 and metal ion activation of the well-known alpha-type PLD from cabbage (PLD alpha). With mixed micelles containing phosphatidyl-p-nitrophenol as substrate, PIP2-concentrations in the nanomolar range are able to activate the enzyme in addition to the essential Ca2+ activation. Mg2+ ions are able to replace Ca2+ ions but they do not activate PLDa. Rather, they abolish the activation of the enzyme by Ca2+ ions in the absence, but not in the presence, of PIP2. The presence of PIP2 causes a shift in the pH optimum of PLDa activity to the acidic range. Employing fluorescence measurements and replacing Ca2+ by Tb3+ 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 beta-sheets consisting of four beta-strands each and loop regions that harbor two Ca2+ binding sites. Based on the findings of the MD simulation, one of the bound Ca2+ ions is coordinated by five amino acid residues. The second Ca2+ ion induces a loop movement upon its binding to three amino acid residues. Docking studies with PIP2 reveal, in addition to the previously postulated PIP2-binding site in the middle of the beta-sheet structure, another PIP2-binding site near the two Ca2 ions, which is in accordance with the experimental interplay of PIP2, Ca2+ and Mg2+ ions.
- 出版日期2017-6
