Allosteric Activation of Phosphatidylinositol-Specific Phospholipase C:  Specific Phospholipid Binding Anchors the Enzyme to the Interface

Abstract

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al. (1997) Biochemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal with PC or PE interfaces. NMR experiments (TRNOE and 31P line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles. These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC6PC and diC7PC, respectively. TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly. The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC8PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or 31P NMR line widths. The 3.6-fold activation produced by polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents. This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure.

The Cation-π Box Is a Specific Phosphatidylcholine Membrane Targeting Motif

“…In so doing they provide a specific recognition motif for the head- groups of zwitterioinic phospholipids. Bacillus PI-PLC is activated by PC (10), in large part by enhancing vesicle binding (25). The G helix region of Bacillus PI-PLCs has several Tyr residues that could form a sandwich or cage around a choline group (22,28), but how and whether PC binds to this box was unclear.…”

“…Similarly, proteins specific to the anionic phospholipid phosphatidylserine (PS) 2 may coordinate Ca 2ϩ ions for PS binding using conserved loop motifs as observed in annexin V (5) or directly bind PS via C2 domains as in coagulation factor V (6) and lactadherin (7) where polar side chains allow specific recognition of PS. The affinity of the proteins for the membrane may be further modulated by insertion of hydrophobic and aromatic amino acids into the bilayer (5,6,8) and/or multivalent interactions (3) via domain repeats (9) or specificity for more than one type of lipid (9,10).…”

“…A small amount of background signal from pure buffer solution or buffer with different concentrations of diC 7 PC was sub-tracted from the control and sample intensities. The dependence of (I Ϫ I 0 )/I 0 on diC 7 PC concentrations reflects protein binding affinity for that short chain lipid (10,28).…”

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

“…Around the barrel rim, there are regions where hydrophobic side chains are exposed to solvent. The structural similarity of the bacterial PI-PLC to the mammalian catalytic domain and similar kinetic features (12)(13)(14) suggest that the smaller enzyme can provide a simple model for understanding how different interfaces modulate catalysis.Previous work has shown that the PI-PLC from Bacillus cereus or Bacillus thuringiensis not only prefers micellar to monomeric substrate (15) but is also activated by binding to phosphatidylcholine (PC) interfaces (2,16,17). The binding has an allosteric effect on the enzyme, since it increases k cat and decreases K m for hydrolysis of the soluble substrate cIP.…”

“…Previous work has shown that the PI-PLC from Bacillus cereus or Bacillus thuringiensis not only prefers micellar to monomeric substrate (15) but is also activated by binding to phosphatidylcholine (PC) interfaces (2,16,17). The binding has an allosteric effect on the enzyme, since it increases k cat and decreases K m for hydrolysis of the soluble substrate cIP.…”