Cross-linking phosphatidylinositol-specific phospholipase C traps two activating phosphatidylcholine molecules on the enzyme

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC(7)PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC(7)PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (betaalpha)(8)-barrel protein. The higher activity of the native protein cross-linked in the presence of diC(7)PC correlated with an increased affinity of the protein for two diC(7)PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC(7)PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.