Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

We monitored the action of phospholipase A₂ (PLA₂) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA₂ molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA₂. The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 μm²/s on the L-DPPC and 4.9 μm²/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to ≈0.2 μm²/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.     

Structural characteristics of hydrolysates of proteins from extracted sunflower flour at the air-water interface

The structural and topographical characteristics of a sunflower protein isolate (SPI) and its hydrolysates at different degrees of hydrolysis (DH = 5.62%, 23.5%, and 46.3%) spread at the air-water interface at pH 7 and 20 degrees C were determined from pi-A isotherms coupled with Brewster angle microscopy (BAM). The structural characteristics of SP hydrolysate spread monolayers depend on the degree of hydrolysis. We observed a significant shift of the pi-A(APPARENT) isotherms toward lower molecular areas as the degree of hydrolysis (DH) increased. This phenomenon was attributed to spreading of the protein at the interface, especially at DH 46.3%. A change in the monolayer structure was observed at a surface pressure of 12-15 mN/m. At a microscopic level, the heterogeneous monolayer structures visualized near the monolayer collapse and during the monolayer expansion proved the existence of large regions of protein aggregates. Reflectivity increased with surface pressure and was a maximum at the monolayer collapse. The monolayer thickness decreased as the degree of hydrolysis increased. These phenomena explain the poor functional properties for the formation and stabilization of a dispersion (emulsion or foam) of protein hydrolysates at high degrees of hydrolysis.     

Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A2: a 13C NMR study

A new approach to study phospholipase A2 mediated hydrolysis of phospholipid vesicles, using 13C NMR spectroscopy, is described. [13C]Carbonyl-enriched dipalmitoylphosphatidylcholine (DPPC) incorporated into nonhydrolyzable ether-linked phospholipid bilayers was hydrolyzed by phospholipase A2 (Crotalus adamanteus). The 13C-labeled carboxyl/carbonyl peaks from the products [lyso-1-palmitoylphosphatidylcholine (LPPC) and palmitic acid (PA)] were well separated from the substrate carbonyl peaks. The progress of the reaction was monitored from decreases in the DPPC carbonyl peak intensities and increases in the product peak intensities. DPPC peak intensity changes showed that only the sn-2 ester bond of DPPC on the outer monolayer of the vesicle was hydrolyzed. Most, but not all, of the DPPC in the outer monolayer was hydrolyzed after 18-24 h. There was no movement of phospholipid from the inner to the outer monolayer over the long time periods (18-24 h) examined. On the basis of chemical shift measurements of the product carbonyl peaks, it was determined that, at all times during the hydrolysis reaction, the LPPC was present only in the outer monolayer of the bilayer and the PA was bound to the bilayer and was approximately 50% ionized at pH approximately 7.2. Bovine serum albumin extracted most of the LPPC and PA from the product vesicles, as revealed by chemical shift changes after addition of the protein. The capability of 13C NMR spectroscopy to elucidate key structural features without the use of either shift reagents or separation procedures which may alter the reaction equilibrium makes it an attractive method to study this enzymatic process.     

Dipalmitoyl-phosphatidylcholine/phospholipase D interactions investigated with polarization-modulated infrared reflection absorption spectroscopy

The hydrolysis of 1,2-dipalmitoylphosphatidylcholine (DPPC) catalyzed by Streptomyces chromofuscus phospholipase D (PLD) has been investigated using monolayer techniques and polarization-modulated infrared absorption reflection spectroscopy. The spectroscopic analysis of the phosphate groups provides a quantitative estimation of the hydrolysis yield. The hydrolysis kinetics was investigated in dependence on the phase state of the lipid monolayer. It was found that PLD exhibits maximum activity in the liquid-expanded phase, whereas PLA2 has its activity maximum in the two-phase region. A lag phase was observed in all experiments indicating that small amounts of the hydrolysis product 1,2-dipalmitoylphosphatidic acid (DPPA) are needed for initiating the fast hydrolysis reaction. Higher concentrations of DPPA inhibit the hydrolysis. The critical inhibition concentration of DPPA is a function of the monolayer pressure.     

Dissection of the steps of phospholipase C beta 2 activity that are enhanced by G beta gamma subunits

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate in a two step reaction that involves a cyclic intermediate. The PLCbetafamily are activated by both the alpha and betagamma subunits of heterotrimeric G proteins. To determine which catalytic step is affected by Gbetagamma subunits, we compared the change in PLCbeta(2) activity catalysis toward monomeric short-chain phosphatidylinositol (PI) substrates and monomeric water-soluble cyclic inositol phosphates as well as long-chain PI in bilayer and micellar interfaces in the absence and presence of Gbetagammasubunits. Unlike other PLC enzymes, no cyclic products were detected for either wild-type PLCbeta(2) or a chimeric protein composed of the PH domain of PLCbeta(2) and the catalytic domain of PLCdelta(1). Using cIP as a substrate to examine the second step of the reaction, we found that the presence of Gbetagamma subunits stimulated this step by a higher level than that for the overall reaction (k(cat) 1.5-fold (cIP) as opposed to 1.20-fold for soluble diC(4)PI). Detergents above their CMC can generate the same kinetic activation of PLCbeta(2) as Gbetagamma, suggesting that hydrophobic compounds stabilize the activated state of the enzyme. The most pronounced effect of Gbetagamma is that it relieves competitive product inhibition. Taken together, our results show that activation of PLCbeta(2) occurs through enhancement in the catalytic rate of hydrolysis of the cyclic intermediate and increased product release, and that hydrophobic interactions play a key role.     

Deletion of the helical motif in the intestinal fatty acid-binding protein reduces its interactions with membrane monolayers: Brewster angle microscopy, IR reflection-absorption spectroscopy, and surface pressure studies

Intestinal fatty acid binding protein (IFABP) appears to interact directly with membranes during fatty acid transfer [Hsu, K. T., and Storch, J. (1996) J. Biol. Chem. 271, 13317-13323]. The largely alpha-helical "portal" domain of IFABP was critical for these protein--membrane interactions. In the present studies, the binding of IFABP and a helixless variant of IFABP (IFABP-HL) to acidic monolayers of 1,2-dimyristoylphosphatidic acid (DMPA) has been monitored by surface pressure measurements, Brewster angle microscopy (BAM), and infrared reflection-absorption spectroscopy (IRRAS). Protein adsorption to DMPA exhibited a two phase kinetic process consisting of an initial slow phase, arising from protein binding to the monolayer and/or direct interfacial adsorption, and a more rapid phase that parallels formation of lipid-containing domains. IFABP exhibited more rapid changes in both phases than IFABP-HL. The second phase was absent when IFABP interacted with zwitterionic monolayers of 1,2-dipalmitoylphosphatidylcholine, revealing the important role of electrostatics at this stage. BAM images of DMPA monolayers with either protein revealed the formation of domains leading eventually to rigid films. Domains of DMPA/IFABP-HL formed more slowly and were less rigid than with the wild-type protein. Overall, the IRRAS studies revealed a protein-induced conformational ordering of the lipid acyl chains with a substantially stronger ordering effect induced by IFABP. The physical measurements thus suggested differing degrees of direct interaction between the proteins and DMPA monolayers with the IFABP/DMPA interaction being somewhat stronger. These data provide a molecular structure rationale for previous kinetic measurements indicating that the helical domain is essential for a collision-based mechanism of fatty acid transfer to phospholipid membranes [Corsico, B., Cistola, D. P., Frieden, C. and Storch, J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 12174-12178].     

Incorporation of beta-lactoglobulin in a lipid/porphyrin monolayer at the air--water interface

A catanionic lipid/porphyrin monolayer was formed at the air-water interface by the tetra-anionic porphyrin, tetra-sodium-meso-tetra(4-sulfonatophenyl)porphine (TSPP), mixed with the cationic lipid dioctadecyldimethylammonium bromide (DODAB) in a 1:4 molar ratio. This binary mixture (TSPP/4DODAB) was used as the incorporation matrix of beta-lactoglobulin (betaLG). Binary and ternary systems (TSPP/4DODAB/zbetaLG, where z stands for the number of protein residues per TSPP) were characterized by surface pressure versus area (pi-A) measurements and by Brewster angle microscopy (BAM) observation at the air-water interface. Pi-A measurements and BAM images show that protein is incorporated in the expanded regime of the monolayer and is gradually expelled upon compression at high surface pressures. The successive compression-expansion cycles indicate that the protein under adsorbed to the floating film is reincorporated after the expansion of the monolayer. At low subphase pH, TSPP tends to aggregate decreasing the interaction with DODAB molecules. Electrostatic and hydrophobic interactions are responsible for the presence of betaLG at the interfacial film.     

Crystal structure of BamD: an essential component of the β-Barrel assembly machinery of gram-negative bacteria

Folding and insertion of integral β-barrel proteins in the outer membrane (OM) is an essential process for Gram-negative bacteria that requires the β-barrel assembly machinery (BAM). Efficient OM protein (OMP) folding and insertion appears to require a consensus C-terminal signal in OMPs characterized by terminal F or W residues. The BAM complex is embedded in the OM and, in Escherichia coli, consists of the β-barrel BamA and four lipoproteins BamBCDE. BamA and BamD are broadly distributed across all species of Gram-negative bacteria, whereas the other components are present in only a subset of species. BamA and BamD are also essential for viability, suggesting that these two proteins constitute the functional core of the bacterial BAM complex. Here, we present the crystal structure of BamD from the thermophilic bacteria Rhodothermus marinus refined to 2.15 Å resolution. The protein contains five tetratricopeptide repeats (TPRs) organized into two offset tandems, each capped by a terminal helix. The N-terminal domain contains three TPRs and displays remarkable structural similarity with proteins that recognize targeting signals in extended conformations. The C-terminal domain harbors the remaining two TPRs and previously described mutations that impair binding to other BAM components map to this domain. Therefore, the structure suggests a model where the C-terminal domain provides a scaffold for interaction with BAM components, while the N-terminal domain participates in interaction with the substrates, either recognizing the C-terminal consensus sequence or binding unfolded OMP intermediates.     

Streptomyces chromofuscus phospholipase D interaction with lipidic activators at the air-water interface

The phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme that interacts with membranes to catalyse phosphatidylcholine (PC) transformation. In this work, we focused on the interaction between PLDSc and two lipid activators: a neutral lipid, diacylglycerol (DAG), and an anionic one, phosphatidic acid (PA). DAG is a naturally occurring alcohol, so it is a potent nucleophile for the transphosphatidylation reaction catalysed by PLD. Concerning PA, it is a widely described activator of PLDSc-catalysed hydrolysis of PC.The monolayer technique allowed us to define PLDSc interaction with DAG and PA. In the case of DAG, the results suggest an insertion of PLDSc within the acyl chains of the lipid with an exclusion pressure of approximately 45 mN/m. PLDSc-DAG interaction seemed to occur preferentially with the lipid in the liquid-expanded (LE) phase.PLDSc interaction with PA was found to be more effective at high surface pressures. The overall results obtained with PA show a preferential interaction of the protein with condensed PA domains. No exclusion pressure could be found for PLDSc-PA interaction indicating only superficial interaction with the polar head of this lipid. Brewster angle microscopy (BAM) images were acquired in order to confirm these results and to visualise the patterns induced by PLDSc adsorption.     

Streptomyces chromofuscus phospholipase D interaction with lipidic activators at the air-water interface

The phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme that interacts with membranes to catalyse phosphatidylcholine (PC) transformation. In this work, we focused on the interaction between PLDSc and two lipid activators: a neutral lipid, diacylglycerol (DAG), and an anionic one, phosphatidic acid (PA). DAG is a naturally occurring alcohol, so it is a potent nucleophile for the transphosphatidylation reaction catalysed by PLD. Concerning PA, it is a widely described activator of PLDSc-catalysed hydrolysis of PC. The monolayer technique allowed us to define PLDSc interaction with DAG and PA. In the case of DAG, the results suggest an insertion of PLDSc within the acyl chains of the lipid with an exclusion pressure of approximately 45 mN/m. PLDSc-DAG interaction seemed to occur preferentially with the lipid in the liquid-expanded (LE) phase. PLDSc interaction with PA was found to be more effective at high surface pressures. The overall results obtained with PA show a preferential interaction of the protein with condensed PA domains. No exclusion pressure could be found for PLDSc-PA interaction indicating only superficial interaction with the polar head of this lipid. Brewster angle microscopy (BAM) images were acquired in order to confirm these results and to visualise the patterns induced by PLDSc adsorption.     

Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.     

The TPR domain of BepA is required for productive interaction with substrate proteins and the β‐barrel assembly machinery complex

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N‐terminal protease domain and a C‐terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone‐like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β‐barrel assembly machinery (BAM) driving integration of β‐barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site‐directed in vivo photo‐cross‐linking was used to map the protein–protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.     

Interfacial regulation of bacterial sphingomyelinase activity

The objective of this study was to define how the quality of the buffer/membrane interface influences the activity of bacterial sphingomyelinase acting at the interface. The enzyme reaction was carried out in a zero-order trough using a surface barostat. This approach allowed for proper control of the physico-chemical properties of the substrate molecules. Since the molecular area of ceramide is smaller than that of sphingomyelin, the hydrolysis reaction could be followed `on-line' from the monolayer area decrease at constant surface pressure. The hydrolysis reaction could be divided into two separate phases, the first being the lag-phase (time between enzyme addition and commencement of the monolayer area change), and the second phase being the actual hydrolysis reaction (from which a maximal degradation rate could be determined). The activity of sphingomyelinase (Staphylococcus aureus) toward bovine brain sphingomyelin (bb-SM) was markedly enhanced by Mg²⁺ (maximal activation at 5 mM). Mg²⁺ also influenced the lag-phase of the reaction (the lag-time increased markedly when the Mg²⁺ concentration decreased below 1 mM). Saturated sphingomyelins (bb-SM and N-palmitoyl sphingomyelin [N-P-SM]) were more slowly degraded than the mono-unsaturated N-oleoyl sphingomyelin (N-O-SM). Both bb-SM and N-P-SM monolayers underwent a phase-transition at room temperature, whereas the N-O-SM monolayer did not. The phase-transition (liquid-expanded to liquid-condensed) was observed to greatly increase the lag-time of the hydrolysis reaction. The activity of sphingomyelinase was also sensitive to the lateral surface pressure of the monolayer membrane. Maximal degradation rate was achieved at 20 mN/m (with bb-SM, 30°C); above this pressure the lag-time of the reaction increased sharply. The inclusion of 4 mol% of cholesterol into a [³H]sphingomyelin monolayer markedly increased the extent of [³H]sphingomyelin degradation, and shortened the lag-time of the reaction. The inclusion of 10 mol% of zwitterionic or negatively charged phospholipids to the [³H]sphingomyelin monolayer did not affect the sphingomyelinase reaction significantly. In conclusion, this study has demonstrated that the physico-chemical properties of the substrate molecules have a dominating influence on the activity of a bacterial sphingomyelinase acting at the buffer/membrane interface.     

The TRPM7 channel is inactivated by PIP(2) hydrolysis

TRPM7 (ChaK1, TRP-PLIK, LTRPC7) is a ubiquitous, calcium-permeant ion channel that is unique in being both an ion channel and a serine/threonine kinase. The kinase domain of TRPM7 directly associates with the C2 domain of phospholipase C (PLC). Here, we show that in native cardiac cells and heterologous expression systems, G alpha q-linked receptors or tyrosine kinase receptors that activate PLC potently inhibit channel activity. Numerous experimental approaches demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP(2)), the substrate of PLC, is a key regulator of TRPM7. We conclude that receptor-mediated activation of PLC results in the hydrolysis of localized PIP(2), leading to inactivation of the TRPM7 channel.     

Presence of SH2 domains of phospholipase C gamma 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor.

src homology region 2 and 3 (SH2 and SH3) domains are conserved noncatalytic regions originally described in cytoplasmic tyrosine kinases and subsequently identified in phospholipase C gamma 1 (PLC gamma 1), GTPase-activating protein of ras, and other signaling proteins. Although numerous studies indicate that SH2 domains promote protein-protein interactions by specific binding to tyrosine phosphorylated proteins, the function of SH3 domains is not known. The SH2 domain of PLC gamma 1 binds to certain tyrosine-phosphorylated growth factor receptors, and following phosphorylation on Tyr783 the enzymatic activity of PLC gamma 1 is enhanced, leading to phosphatidylinositol hydrolysis. To determine the functional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in Escherichia coli PLC gamma 1 constructs encoding the region containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjected to quantitative phosphorylation by the epidermal growth factor receptor (EGFR). Analysis of the kinetics of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanced for substrates containing SH2 domains: from S0.5 (average apparent Km) of 110 microM to S0.5 of 20 microM with the addition of a single SH2 domain and S0.5 of 3-4 microM for mutants containing two SH2 domains. The presence of the SH3 domain did not influence the apparent Km of substrate phosphorylation. These results demonstrate that the presence of the SH2 domain in PLC gamma 1 lowers the apparent Km (increases the affinity) of substrate phosphorylation by the EGFR, thereby facilitating PLC gamma 1 phosphorylation and activation.     

Lateral electrical conduction along a phosphatidylcholine monolayer

Lateral electrical conduction due to lipid-monolayers spread on the surface of pure water was observed under both d.c. and a.c. electrical fields. An apparent specific electrical conductivity is evaluated as high as approximately equal to 4.10(-2) mho/cm for the monolayer-water system of L-DPPC at 25 degrees C. Arrhenius plots of the apparent conductance show a deflection at a temperature corresponding to a crystalline-to-fluid phase transition of the surface monolayer. From the magnitude and temperature dependence of conductance and a comparison of results with those obtained by use of deuterated water, it is concluded that enhanced protonic conduction mediated by a network consisted of polar head groups of phosphatidylcholines and water molecules may be brought about near the lipid/water interface.     

Phospholipase C isoforms are localized at the cleavage furrow during cytokinesis

It has recently been demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP2) is localized at the cleavage furrow in dividing cells and its hydrolysis is required for complete cytokinesis, suggesting a pivotal role of PIP2 in cytokinesis. Here, we report that at least three mammalian isoforms of phosphoinositide-specific phospholipase C (PLC), PLCdelta1, PLCdelta3 and PLCbeta1, are localized to the cleavage furrow during cytokinesis. Targeting of the delta1 isoform to the furrow depends on the specific interaction between the PH domain and PIP2 in the plasma membrane. The necessity of active PLC in animal cell cytokinesis was confirmed using the specific inhibitors for PIP2 hydrolysis. These results support the model that activation of selected PLC isoforms at the cleavage furrow controls progression of cytokinesis through regulation of PIP2 levels: induction of the cleavage furrow by a contractile ring consisting of actomyosin is regulated by PIP2-dependent actin-binding proteins and formation of specific lipid domains required for membrane separation is affected by alterations in the lipid composition of the furrow.     

Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.