Cloning, expression and functional characterization of the C2 domain from tomato phospholipase Dα

C2 domains exist as highly conserved N-terminal or C-terminal calcium- and lipid-binding motifs comprising nearly 130 amino acids, responsible for recruiting proteins to the membrane during signal transduction. In this study, the sequence corresponding to the N-terminal 164 amino acids of a full length cDNA of phospholipase Dα from tomato fruit was cloned in pET28(b) vector and expressed in E. coli as a His-tagged protein. Recombinant C2 domain showed micromolar affinity towards Ca++ with a maximum of 2 high affinity binding sites. Interaction of C2 domain with synthetic unilamellar vesicles, evaluated by protein- lipid fluorescence resonance energy transfer, showed maximum affinity towards phosphatidic acid, and virtually no binding with phosphatidylcholine. The binding towards phosphoinositides was reduced with increasing degree of phosphorylation. Acid- and chaotropic salt- titrations indicated an electrostatic, rather than a hydrophobic mode of interaction between C2 domain and the phospholipid vesicles. Conformational analyses of the recombinant C2 domain showed a much longer calcium binding loop region, a far less electropositive phosphoinositide-binding region, unique calcium binding pockets with high electro-negativity, and other features that are distinct from the typical C2 domains of phospholipase A2 and Protein kinase C α, signifying the uniqueness of Phospholipase Dα in fruit developmental events.