title: Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of α-type plant phospholipase D.
abstract: Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca2+binding sites. In vitro, the predominantly expressed α-type PLDs need 20-100 mM CaCl2for optimum activity, whereas the essential activator of β- or γ-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP2), plays a secondary role. In the present paper, we have studied the interplay between PIP2and metal ion activation of the well-known α-type PLD from cabbage (PLDα). 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 PLDα. Rather, they abolish the activation of the enzyme by Ca2+ions in the absence, but not in the presence, of PIP2. The presence of PIP2causes a shift in the pH optimum of PLDα 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 β-sheets consisting of four β-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 PIP2reveal, in addition to the previously postulated PIP2-binding site in the middle of the β-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.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Dreßler
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Michel
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: Thondorf
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Mansfeld
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Golbik
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany. Electronic address: renate.ulbrich-hofmann@biochemtech.uni-halle.de.
authors.forename: Renate
authors.initials: R
authors.lastname: Ulbrich-Hofmann
mesh_terms: D001665:Binding Sites; D001937:Brassica / Q000201:enzymology*; D002118:Calcium / Q000737:chemistry; D007477:Ions / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D010739:Phospholipase D / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013725:Terbium / Q000737:chemistry
chemical_list: D007477:Ions; D008670:Metals; D019269:Phosphatidylinositol 4,5-Diphosphate; D010940:Plant Proteins; D013725:Terbium; D010739:Phospholipase D; D008274:Magnesium; D002118:Calcium
keywords: Activity; Brassica oleracea convar. capitata var. alba L.; Brassicaceae; C2 domain; Calcium; Fluorescence; Homology modelling; Mixed micelles; Molecular docking; PIP2; Phospholipase D