Search and Subvert: Minimalist Bacterial Phosphatidylinositol-Specific Phospholipase C Enzymes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes from Gram-positive bacteria are secreted virulence factors that aid in downregulating host immunity. These PI-PLCs are minimalist peripheral membrane enzymes with a distorted (ßα)8 TIM barrel fold offering a conserved and stable scaffold for the conserved catalytic amino acids while membrane recognition is achieved mostly through variable loops. Decades of experimental and computational research on these enzymes have revealed the subtle interplay between molecular mechanisms of catalysis and membrane binding, leading to a semiquantitative model for how they find, bind, and cleave their respective substrates on host cell membranes. Variations in sequence and structure of their membrane binding sites may correlate with how enzymes from different Gram-positive bacteria search for their particular targets on the membrane. Detailed molecular characterization of protein-lipid interactions have been aided by cutting-edge methods ranging from 31P field-cycling NMR relaxometry to monitor protein-induced changes in phospholipid dynamics to molecular dynamics simulations to elucidate the roles of electrostatic and cation-π interactions in lipid binding to single molecule fluorescence measurements of dynamic interactions between PI-PLCs and vesicles. This toolkit is readily applicable to other peripheral membrane proteins including orthologues in Gram-negative bacteria and more recently discovered eukaryotic minimalist PI-PLCs.

Dimer structure of an interfacially impaired phosphatidylinositol-specific phospholipase C.

The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8A 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 --> 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 membrane-partitioning residues.

Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes.

The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.

A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol.

The Toxoplasma gondii phosphoinositide-specific phospholipase C gene (TgPI-PLC) was cloned, sequenced and expressed in Escherichia coli and its enzymatic characteristics were investigated. TgPI-PLC is present in the genome as a single-copy gene consisting of 22 exons interrupted by 21 introns, and encodes a polypeptide of 1097 amino acids with a predicted molecular mass of 121 kDa. In addition to the conserved catalytic X and Y domains, TgPI-PLC contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When compared with mammalian delta-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31-35% identity over the whole sequence. Recombinant TgPI-PLC, as well as the native enzyme obtained from crude membrane extracts of the parasite, was more active with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. Indirect immunofluorescence analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme localizes in the plasma membrane of the parasites.

Modulation of PI-specific phospholipase C by membrane curvature and molecular order.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus has been assayed on large and small unilamellar vesicles consisting of PI, either pure or in mixtures with other lipids. Vesicle diameter (in the 50-300 nm range) influences PI-PLC activity, enzyme rates increasing with decreasing curvature radii. With sonicated unilamellar vesicles of pure PI, two apparent K(s) values are observed, one in the 0-2 mM concentration range and the other in the 2-12 mM concentration range. The latter ( approximately 4.2 mM) corresponds to previously published values, while the low-concentration K(s) is on the same order of magnitude as the single apparent K(m) value found with large unilamellar liposomes ( approximately 0.30 mM). PI-PLC appears to be very sensitive to bilayer composition. Certain nonsubstrate lipids, e.g., galactosylceramide or cholesterol, inhibit PI-PLC in a dose-dependent way, at least up to 33 mol % in the bilayers, under conditions with a constant PI concentration. Simultaneous measurements of enzyme activity, interfacial enzyme binding, and fluorescence of different probes, on a variety of bilayer compositions, reveal that both the level of enzyme binding and activity decrease with increasing lipid order, as measured by the fluorescence polarization of the hydrophobic probe diphenylhexatriene. In contrast, no correlation is found for enzyme activity with fluorescence changes of probes, e.g., laurdan, that report on phenomena occurring mainly at the lipid-water interface. Sphingomyelin has a dual effect. Up to 40 mol %, it increases PI-PLC activity, with little effect on bilayer molecular order. At higher proportions, the increased lipid chain order causes a decrease in enzyme activity. The same effects are observed for distearoylphosphatidylcholine when added to PI bilayers. These results support the "two-stage model" for binding of PI-PLC to lipid bilayers, and underline the significance of the enzyme partial penetration into the membrane hydrophobic matrix for its catalytic activity.

Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors.

Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) plays a critical role in escape of this human pathogen from host cell vacuoles. Unlike classical bacterial PI-PLCs, the L. monocytogenes enzyme has very weak activity on glycosylphosphatidylinositol (GPI)-anchored proteins. Previous crystal structure analysis has revealed that a small beta-strand (Vb) is present in Bacillus cereus PI-PLC and is absent in the enzyme from L. monocytogenes. This Vb beta-strand in B. cereus PI-PLC forms contacts with the glycan linker of GPI anchors, which presumably increases its activity on GPI-anchored proteins. In this study, we show that, of all known bacterial PI-PLCs, those from listeriae are the only ones that lack the beta-strand. Expression by L. monocytogenes of B. cereus PI-PLC, which has strong activity on GPI-anchored proteins, inhibited bacterial escape from a vacuole and cell-to-cell spread, resulting in greatly reduced virulence in mice. Deletion of the Vb beta-strand from B. cereus PI-PLC abolished its ability to cleave GPI-anchored proteins, decreased its inhibitory effects, and increased its virulence in mice. These results strongly suggest that L. monocytogenes PI-PLC has evolved as an important determinant of L. monocytogenes pathogenesis by absence of the Vb beta-strand, thus leading to greatly reduced activity on GPI-anchored proteins.

X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: insight into the role of calcium and surrounding amino acids in active site geometry and catalysis.

Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thuringiensis to 2.1 A resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca(2+) ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.

Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Deltaplc1, Deltaarg82, and Deltakcs1 deletion strains, but not the Deltaipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation.

Evaluation of a chromogenic medium for identification and differentiation of Listeria monocytogenes in selected foods.

Listeria monocytogenes continues to be a threat to food safety in the United States despite a "zero tolerance" policy. When Listeria species are identified by standard cultural methods, confirmation of L. monocytogenes takes days to complete. RAPID'L.Mono agar, developed by Bio-Rad Laboratories, is a chromogenic medium that differentiates L. monocytogenes from other species of Listeria by a simple color change reaction. Differentiation is based on the specific detection of phosphatidylinositol phospholipase C activity, resulting in a blue colony, and the inability of L. monocytogenes to metabolize xylose, resulting in the absence of a yellow halo. Detection principles of standard method agars, Oxford and PALCAM, are based on the ability of all species of Listeria to hydrolyze esculin. Thus, all species of Listeria have similar colony morphology on these agars, making differentiation of pathogenic L. monocytogenes from other nonhuman pathogens difficult. RAPID'L.Mono agar has been validated with surimi, mixed salad, brie, and processed deli turkey because of the prevalence of L. monocytogenes in these foods. Sensitivity and specificity for this medium was determined to be 99.4 and 100%, respectively. Overall method agreement of RAPID'L.Mono with standard culture methods (U.S. Department of Agriculture/Food Safety and Inspection Service; U.S. Food and Drug Administration/Bacteriological Analytical Manual; and AOAC INTERNATIONAL) was excellent, with enrichment protocols 24 h shorter than those of standard methods.

Identification of a novel class of mammalian phosphoinositol-specific phospholipase C enzymes.

Phosphoinositol (PhoIns)-specific phospholipase C enzymes (PLCs) are central to the inositol lipid signaling pathways and contribute to intracellular Ca2+ release and protein kinase C activation. Five distinct classes of PhoIns-specific PLCs are known to exist in mammals, which are activated by membrane receptor-mediated events. Here we have identified a sixth class of PhoIns-specific PLC with a novel domain structure, which we have termed PLC-eta. Two putative PLC-eta enzymes were identified in humans and in mice. Sequence analysis revealed that residues implicated in substrate binding and catalysis from other PhoIns-specific PLCs are conserved in the novel enzymes. PLC-eta enzymes are most closely related to the PLC-delta class and share a close evolutionary relationship with other PLC isozymes. EST analysis and RT-PCR data suggest that PLC-eta enzymes are expressed in several cell types and, by analogy with other mammalian PhoIns-specific PLCs, are likely to be involved in signal transduction pathways.

Phosphoinositide-specific phospholipase C is involved in cytokinin and gravity responses in the moss Physcomitrella patens.

The phosphoinositide signalling pathway is important in plant responses to extracellular and intracellular signals. To elucidate the physiological functions of phosphoinositide-specific phopspholipase C, PI-PLC, targeted knockout mutants of PpPLC1, a gene encoding a PI-PLC from the moss Physcomitrella patens, were generated via homologous recombination. Protonemal filaments of the plc1 lines show a dramatic reduction in gametophore formation relative to wild type: this was accompanied by a loss of sensitivity to cytokinin. Moreover, plc1 appeared paler than the wild type, the result of an altered differentiation of chloroplasts and reduced chlorophyll levels compared with wild type filaments. In addition, the protonemal filaments of plc1 have a strongly reduced ability to grow negatively gravitropically in the dark. These effects imply a significant role for PpPLC1 in cytokinin signalling and gravitropism.

The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion.

Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274). To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C.

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.

Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E(#) with monodisperse diheptanoylphosphatidylcholine (DC(7)PC) that is distinguishable from the E complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC(7)PC give an apparent dissociation constant of K(2) = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E(#) are distinct from those of the E complex. The aggregation and partitioning behaviors suggest that the acyl chains in E(#) but not in E remain exposed to the aqueous phase. The free (E) and complexed (E(#) and E) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC(7)PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis-Menten complex formed with monodisperse DC(7)PC is likely to be E(#)S or its aggregate rather than the classical monodisperse ES complex.

Phosphatidylinositol-dependent bond between alkaline phosphatase and collagen fibers in the periodontal ligament of rat molars.

Alkaline phosphatase (ALP) is anchored to the outer leaflet of the lipid bilayer via phosphatidylinositol (PI) and ALP activity has been localized in the plasma membrane of numerous tissues. In the periodontal ligament ALP activity is found in the collagen fibers in addition to the plasma membrane of the osteoblasts and fibroblasts. In this study, we examined the distribution of ALP activity in the periodontal ligament of rat molars and also examined whether the bond between ALP and collagen fibers is dependent on PI by using phosphatidylinositol-specific phospholipase C (PI-PLC). ALP activity was distributed in the periodontal ligament. The activity mirrored the distribution of collagen fibers in the periodontal ligament. Cytochemical analysis also demonstrated that ALP activity was located not only in the plasma membrane of fibroblasts, but also in the collagen fiber bundles and fibrils in the periodontal ligament. After treatment with PI-PLC, the loss of ALP activity in the periodontal ligament was observed histochemically, and the loss of ALP activity in the fibroblasts as well as in the collagen fiber bundles and fibrils was observed cytochemically. These results strongly indicate that the bond between ALP and the collagen fibers is also dependent on PI.

Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C.

Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.