Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

Abstract

The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8 Å resolution. The W47A/ W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp 3 Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membranepartitioning residues.Phosphatidylinositol-specific phospholipase C (PI-PLC) 2 enzymes catalyze the specific cleavage of the sn-3-phosphodiester bond in phosphatidylinositol (PI) and phosphoinositides. PI hydrolysis occurs in two sequential reactions (1-3): (i) an intramolecular phosphotransferase reaction at a phospholipid/ aggregate surface to produce diacylglycerol and water-soluble 1,2-cyclic inositol phosphate (cIP) (or cIP x in the case of a phosphoinositide substrate), followed by (ii) a phosphodiesterase reaction where water-soluble cIP is hydrolyzed to inositol-1-phosphate. Since their substrate is presented in an interface, their activity can often be modulated by the composition of the interface. Eukaryotic PI-PLCs, as critical components of phosphatidylinositol-mediated signaling pathways (4, 5), have multiple domains for regulation of enzyme activity both by regulating partitioning to membranes and by allosteric effects on catalysis, and these are often hard to separate. However, the much smaller bacterial PI-PLC enzymes are secreted, single domain proteins whose crystal structures (6 -8) resemble the catalytic domain of PLC␦ 1 , the only mammalian PI-PLC for which there is a structure (9 -11). The molecular topology is that of a distorted TIM barrel. 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...

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

“…B. thuringiensis PI-PLC was labeled with AF488 maleimide at a single Cys residue introduced at Asn 168 (N168C). Asn 168 is on an exterior helix of B. thuringiensis PI-PLC, far from both the proposed protein dimerization interface (14) and the proposed lipid interface (11). N168C labeled with AF488 had a PI cleavage rate of 1600 Ϯ 160 mol min Ϫ1 mg Ϫ1 at 28°C toward 8 mM PI dispersed in 32 mM diC 7 PC micelles, the same value as for wild-type PI-PLC assayed under the same conditions.…”

“…Our current model for kinetic activation of B. thuringiensis PI-PLC, based on the crystal structure of an interfacially impaired mutant protein (14) and extensive mutagenesis of surface residues (15)(16)(17), is that specific binding to PC in the lipid membrane allows key loop residues to penetrate the interface, promoting an enzyme conformation (suggested to be a transient dimer (14,17)) with enhanced catalytic activity. However, in all PI vesicle assay systems examined, when the bulk PI is kept constant but the surface concentration decreases, PI-PLC specific activity decreases dramatically above X PC ϭ 0.50 (12,18).…”

Molecular Determinants for Interfacial Binding and Conformational Change in a Soluble Diacylglycerol Kinase

“…with membranes via hydrophobic interactions involving tryptophan residues (6). These residues are located on the rim of a (␤␣) 8 -barrel flanking the entrance to the active site and function as interfacial anchors that selectively recognize phosphatidylcholine (PtdCho) (7,8). X-ray studies reveal that the active sites of these enzymes are completely formed in solution (9 -13) (7,14).…”

“…These residues are located on the rim of a (␤␣) 8 -barrel flanking the entrance to the active site and function as interfacial anchors that selectively recognize phosphatidylcholine (PtdCho) (7,8). X-ray studies reveal that the active sites of these enzymes are completely formed in solution (9 -13) (7,14). However, phospholipases A and C do undergo subtle conformational changes upon binding to the interface that cooperate in promoting bilayer association and processive catalysis (5,7,(15)(16)(17)(18).…”

“…X-ray studies reveal that the active sites of these enzymes are completely formed in solution (9 -13) (7,14). However, phospholipases A and C do undergo subtle conformational changes upon binding to the interface that cooperate in promoting bilayer association and processive catalysis (5,7,(15)(16)(17)(18). These conformational changes are important for the i-face to form a tight seal with the bilayer to exclude water from the active site channel and allow substrate entry, but appear to have a minimal effect on the configuration of the active site.…”