Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide-specific phospholipase C delta 1.
Phosphatidylinositide-specific phospholipase Cs (PI-PLCs) catalyze the calcium-dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI-PLCs contain divergent regulatory regions, but all share three conserved regions: an N-terminal pleckstrin homology (PH) domain, X, and Y. We report the high-level expression and characterization of a recombinant "catalytic core" of rat PI-PLC delta 1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI-PLC delta delta 1-134, is catalytically active versus phosphatidylinositol 4,5-bisphosphate in deoxycholate micelles with a K(m) of 182 microM and a Vmax of 27 mumol/min/mg. PI-PLC delta delta 1-134 is monomeric and monodisperse as judged by dynamic light scattering. Far-UV CD indicates a structure with approximately 35% alpha-helix. A reversible change in the near-UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI-PLC delta delta 1-134 in the absence of phospholipid. Triclinic crystals of PI-PLC delta delta 1-134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/A3 and on the self-rotation function, there are two PI-PLC delta delta 1-134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.
Grobler
J A
JA
Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0580, USA.
Hurley
J H
JH
D000818:Animals; D002478:Cells, Cultured; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D054801:Phosphoinositide Phospholipase C; D054803:Phospholipase C delta; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats
D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D054803:Phospholipase C delta