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.
Department of Plant Agriculture, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Krishnaraj
K
Tiwari
Gopinadhan
G
Paliyath
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005638:Fruit; D015870:Gene Expression; D005822:Genetic Vectors; D018551:Solanum lycopersicum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015388:Organelles; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D015398:Signal Transduction; D055672:Static Electricity
D018076:DNA, Complementary; D010743:Phospholipids; D010940:Plant Proteins; D011994:Recombinant Proteins; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; D002118:Calcium