Distinct Ca2+ Binding Properties of Novel C2 Domains of Plant Phospholipase Dα and β

• Published in: The Journal of biological chemistry Vol. 275; no. 26; pp. 19700 - 19706
• Main Authors: Zheng, Li, Krishnamoorthi, Ramaswamy, Zolkiewski, Michal, Wang, Xuemin
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 30.06.2000
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M001945200

Of the isoforms of plant phospholipase D (PLD) that have been cloned and characterized, PLDα requires millimolar levels of Ca2+ for optimal activity, whereas PLDβ is most active at micromolar concentrations of Ca2+. Multiple amino acid sequence alignments suggest that PLDα and PLDβ both contain a Ca2+-dependent phospholipid-binding C2 domain near their N termini. In the present study, we expressed and characterized the putative C2 domains of PLDα and PLDβ, designated PLDα C2 and PLDβ C2, by CD spectroscopy, isothermal titration calorimetry, and phospholipid binding assay. Both PLD C2 domains displayed CD spectra consistent with anticipated major β-sheet structures but underwent spectral changes upon binding Ca2+; the magnitude was larger for PLDβ C2. These conformational changes, not shown by any of the previously characterized C2 domains of animal origin, occurred at micromolar Ca2+ concentrations for PLDβ C2 but at millimolar levels of the cation for PLDα C2. PLDβ C2 exhibited three Ca2+-binding sites: one with a dissociation constant (Kd) of 0.8 μm and the other two with a Kd of 24 μm. In contrast, isothermal titration calorimetry data of PLDα C2 were consistent with 1–3 low affinity Ca2+-binding sites withKd in the range of 590–470 μm. The thermodynamics of Ca2+ binding markedly differed for the two C2 domains. Likewise, PLDβ C2 bound phosphatidylcholine (PC), the substrate of PLD, in the presence of submillimolar Ca2+concentrations, whereas PLDα C2 did so only in the presence of millimolar levels of the metal ion. Both C2 domains bound phosphatidylinoistol 4,5-bisphosphate, a regulator of PC hydrolysis by PLD. However, added Ca2+ displaced the bound phosphatidylinoistol 4,5-bisphosphate. Ca2+ and PC binding properties of PLDα C2 and PLDβ C2 follow a trend similar to the Ca2+ requirements of the whole enzymes, PLDα and PLDβ, for PC hydrolysis. Taken together, the results suggest that the C2 domains of PLDα and PLDβ have novel structural features and serve as handles by which Ca2+ differentially regulates the activities of the isoforms.