Activation of Plant Phospholipase Dβ by Phosphatidylinositol 4,5-Bisphosphate: Characterization of Binding Site and Mode of Action

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Abstract
Hydrolysis of phospholipids by plant phospholipase Dβ (PLDβ) requires phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here we show that PLDβ is stimulated by different polyphosphoinositides, among which PI(4,5)P2 is most effective. On the basis of amino acid sequence analysis, PI(4,5)P2 binding assay, and protein engineering studies, we have identified in the catalytic region of PLDβ a new PI(4,5)P2 binding region (PBR1), which is conserved in eukaryotic PLDs. PBR1 is a second domain besides the previously characterized N-terminal C2 domain of PLDβ which also binds PI(4,5)P2. Submillimolar levels of calcium ions, while inhibiting PI(4,5)P2 binding by the C2 domain, enhanced the affinity of PBR1 for that phosphoinositide. Substrate binding by PLDβ was promoted by PI(4,5)P2-bound PBR1. Isolated, recombinant PBR1 bound PI(4,5)P2 specifically and in a saturable manner. Deletion of PBR1 from PLDβ or mutation of the conserved basic amino acid residues in PBR1 (K437G/K440G) abolished the enzymatic activity. Circular dichroism spectroscopy revealed a conformational change caused by PI(4,5)P2 binding to the catalytic region of PLD. The conformational change apparently helps in the recruitment of the substrate to the active site of the enzyme. The results taken together allow us to describe an anchorage-scooting model for the synergistic activation of PLDβ by PI(4,5)P2 and Ca2+.