Activation of plant phospholipase Dβ by phosphatidylinositol 4,5-bisphosphate:: Characterization of binding site and mode of action

Hydrolysis of phospholipids by plant phospholipase Dbeta (PLDbeta) requires phosphatidylinositol 4,5-bisphosphate [PI(4,5)P-2]. Here we show that PLDbeta is stimulated by different polyphosphoinositides, among which PI(4,5)P-2 is most effective. On the basis of amino acid sequence analysis, PI(,4,5)P-2 binding assay, and protein engineering studies, we have identified in the catalytic region of PLDbeta a new PI(4,5)P2 P-2 binding region (PBR1), which is conserved in eukaryotic PLDs. PBR1 is a second domain besides the previously characterized N-terminal C2 domain of PLDbeta which also binds PI(4,5)P-2. Submillimolar levels of calcium ions, while inhibiting PI(4,5)P-2 binding by the C2 domain, enhanced the affinity of PBR1 for that phosphoinositide. Substrate binding by PLDbeta was promoted by PI(4,5)P-2-bound PBR1. Isolated, recombinant PBR1 bound PI(4,5)P-2 specifically and in a saturable manner. Deletion of PBR1 from PLDbeta or mutation of the conserved basic araino acid residues in PBR1 (K437G/K440G) abolished the enzymatic activity. Circular dichroism spectroscopy revealed a conformational change caused by PI(4,5)P-2 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 to-ether allow us to describe an anchorage-scooting model for the synergistic activation of PLDbeta by PI(4,5)P-2 and Ca2+.