Optimizing the interfacial binding and activity of a bacterial phosphatidylinositol-specific phospholipase C

The phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis can be activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers. This activation corresponds with partial insertion into the interface of two tryptophans, Trp-47 in helix B and Trp-242 in a loop, in the rim of the alphabeta-barrel. Both W47A and W242A have much weaker binding to interfaces and considerably lower kinetic interfacial activation. Tryptophan rescue mutagenesis, reinsertion of a tryptophan at a different place in helix B in the W47A mutant or in the loop (residues 232-244) of the W242A mutant, has been used to determine the importance and orientation of a tryptophan in these two structural features. Phosphotransferase and phosphodiesterase assays, and binding to phosphatidylcholine vesicles were used to assess both orientation and position of tryptophans needed for interfacial activity. Of the helix B double mutants, only one mutant, I43W/W47A, has tryptophan in the same orientation as Trp-47. I43W/W47A shows recovery of phosphatidylinositol-specific phospholipase C (PC) activation of d-myo-inositol 1,2-cyclic phosphate hydrolysis. However, the specific activity toward phosphatidylinositol is still lower than wild type enzyme and high activity with phosphatidylinositol solubilized in 30% isopropyl alcohol (a hallmark of the native enzyme) is lost. Reinserting a tryptophan at several positions in the loop composed of residues 232-244 partially recovers PC activation and affinity of the enzyme for lipid interfaces as well as activation by isopropyl alcohol. G238W/W242A shows an enhanced activation and affinity for PC interfaces above that of wild type. These results provide constraints on how this bacterial phosphatidylinositol-specific phospholipase C binds to activating PC interfaces.     

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.     

The spider toxin omega-Aga IIIA defines a high affinity site on neuronal high voltage-activated calcium channels

The spider toxin omega-agatoxin IIIA (omega-Aga-IIIA) is a potent inhibitor of high voltage-activated calcium currents in the mammalian brain. To establish the biochemical parameters governing its action, we radiolabeled the toxin and examined its binding to native and recombinant calcium channels. In experiments with purified rat synaptosomal membranes, both kinetic and equilibrium data demonstrate one-to-one binding of omega-Aga-IIIA to a single population of high affinity sites, with K(d) = approximately 9 pm and B(max) = approximately 1.4 pmol/mg protein. Partial inhibition of omega-Aga-IIIA binding by omega-conotoxins GVIA, MVIIA, and MVIIC identifies N and P/Q channels as components of this population. omega-Aga-IIIA binds to recombinant alpha(1B) and alpha(1E) calcium channels with a similar high affinity (K(d) = approximately 5-9 pm) in apparent one-to-one fashion. Results from recombinant alpha(1B) binding experiments demonstrate virtually identical B(max) values for omega-Aga-IIIA and omega-conotoxin MVIIA, providing further evidence for a one-to-one stoichiometry of agatoxin binding to calcium channels. The combined evidence suggests that omega-Aga-IIIA defines a unique, high affinity binding site on N-, P/Q-, and R-type calcium channels.     

Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.     

The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.     

On the relationship between the dual specificity of the bovine brain phosphatidylinositol transfer protein and membrane phosphatidylinositol levels

The phosphatidylinositol transfer protein from bovine brain has a remarkable specificity pattern with a distinct preference for phosphatidylinositol (PI) and a low affinity for phosphatidylcholine (PC). In this study we have determined the affinity of PI-transfer protein for PI relative to that for PC by measuring the binding of the fluorescent pyrene-labeled analogs of these phospholipids. From competition binding experiments it was estimated that the transfer protein has a 16-fold higher affinity for PI than for PC. This relative affinity together with the relative abundance of PI and PC, determines what proportion of the protein contains PI (e.g. 65% of the PI-transfer protein in the case of bovine brain). From measuring lipid transfer between donor vesicles consisting of equimolar amounts of PC and PI, and an excess of acceptor vesicles consisting of various ratios of PC and PI, we have observed that the relative rates of the PI-transfer protein-mediated transfer of PI and PC varies between 5 and 20. Kinetic analysis has indicated that PI-transfer protein carrying a PI molecule has different kinetic properties than the PI-transfer protein carrying a PC molecule. It will be discussed that because of the dual specificity, PI-transfer protein is ideally suited for maintaining PI levels in intracellular membranes.     

Phosphorylation of glycosyl-phosphatidylinositol by phosphatidylinositol 3-kinase changes its properties as a substrate for phospholipases

Phosphatidylinositol 3-kinases (PI3K) phosphorylate the 3-position of the inositol ring of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate. It is not clear whether PI3K can phosphorylate the inositol group in other biomolecules. We sought to determine whether PI3K was able to use glycosyl-phosphatidylinositol (GPI) as a substrate. This phospholipid may exist either in free form (GPIfree) or forming a lipid anchor (GPIanchor) for the attachment of extracellular proteins to the plasma membrane. We demonstrate the specific PI3K-mediated phosphorylation of the inositol 3-hydroxyl group within both types of GPI by incubating this phospholipid with immunoprecipitated PI3K. The phosphorylated product behaves in HPLC as a derivative of a PI3K lipid product. To our knowledge, this is the first demonstration that PI3K uses lipid substrates other than phosphoinositides. Further, we show that this has potential functional consequences. When GPIfree is phosphorylated, it becomes a poorer substrate for GPI-specific phospholipase D, but a better substrate for phosphatidylinositol-specific phospholipase C. These phosphorylation events may constitute the basis of a previously undescribed signal transduction mechanism.     

A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔG_el below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought.     

Phosphatidic acid regulates the affinity of the murine phosphatidylinositol 4-phosphate 5-kinase-Ibeta for phosphatidylinositol-4-phosphate

Type I phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) catalyzes the phosphorylation of phosphatidylinositol 4 phosphate [PI(4)P] at carbon 5, producing phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Phosphatidic acid (PA) activates PI4P5K in vitro and plays a central role in the activation of PIP5K pathways in vivo. This report demonstrates that actin fiber formation in murine fibroblasts involves PA activation of PIP5Ks and defines biochemical interactions between PA and the PIP5Ks. Inhibition of phospholipase D production of PA results in the loss of actin fibers. Overexpression of the beta isoform of the type I murine phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta) maintains actin fiber structure in the face of phospholipase D inhibition. PA activates mPIP5K-Ibeta by direct binding to mPIP5K-Ibeta through both electrostatic and hydrophobic interactions, with the fatty acid acyl chain length and degree of saturation acting as critical determinants of binding and activation. Furthermore, kinetic analysis suggests that phosphorylation of the PI(4)P substrate does not follow classical Michaelis-Menten kinetics. Instead, the kinetic data are consistent with a model in which mPIP5K-Ibeta initially binds to the lipid micelle and subsequently binds the PI(4)P substrate. In addition, the kinetics indicate substrate inhibition, suggesting that mPIP5K-Ibeta contains an inhibitory PI(4)P-binding site. These results suggest a model in which mPIP5K-Ibeta is surrounded by PI(4)P, but is unable to catalyze its conversion to PI(4,5)P2 unless PA is bound.     

Glucosylceramide,a neutral glycosphingolipid anticoagulant cofactor,enhances the interaction of human- and bovine-activated protein C with negatively charged phospholipid vesicles

The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with a K(d) (app) = 16 micro g/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with a K(d) (app) = 3 micro g/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of approximately 63 and approximately 64 A were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming kappa(2) = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent K(d) of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6-7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal gamma-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids.     

Investigating the interfacial binding of bacterial phosphatidylinositol-specific phospholipase C

The interactions of PI-PLC with nonsubstrate zwitterionic [phosphatidylcholine (PC)] and anionic [phosphatidylmethanol (PMe), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid] interfaces that affect the catalytic activity of PI-PLC have been examined. PI-PLC binding is strongly coupled to vesicle curvature and is tighter at acidic pH for all of the phospholipids examined. PI-PLC binds to small unilamellar vesicles (SUVs) of anionic lipids with much higher affinity (K(d) is 0.01-0.07 microM for a site consisting of n = 100 +/- 25 lipids when analyzed with a Langmuir adsorption isotherm) than to zwitterionic PC SUVs (K(d) is 5-20 microM and n = 8 +/- 3). The binding to PC surfaces is dominated by hydrophobic interactions, while binding to anionic surfaces is dominated by electrostatic interactions. The contributions of specific cationic side chains and hydrophobic groups at the rim of the alpha beta-barrel to zwitterionic and anionic vesicle binding have been assessed with mutagenesis. The results are used to explain how PC activates the enzyme for both phosphotransferase and cyclic phosphodiesterase activities.     

PDZ domains of Par-3 as potential phosphoinositide signaling integrators

Multiple PDZ domain scaffold protein Par-3 and phosphoinositides (PIPs) are required for polarity in diverse cell types. We show that the second PDZ domain of Par-3 binds to phosphatidylinositol (PI) lipid membranes with high affinity. We further demonstrate that a large subset of PDZ domains in mammalian genomes are capable of binding to PI lipid membranes, indicating that lipid binding is the second most prevalent interaction mode of PDZ domains known to date. The biochemical and structural basis of Par-3 PDZ2-mediated membrane interaction is characterized in detail. The membrane binding capacity of Par-3 PDZ2 is critical for epithelial cell polarization. Interestingly, the lipid phosphatase PTEN directly binds to the third PDZ domain of Par-3. The concatenation of the PIP-binding PDZ2 and the lipid phosphatase PTEN-binding PDZ3 endows Par-3 as an ideal scaffold protein for integrating PIP signaling events during cellular polarization.     

Affinity analysis of non-steady-state data obtained under mass transport limited conditions using BIAcore technology

Binding data obtained with Biacore instrumentation is often evaluated using a kinetic transport model where reaction rate constants and a mass transport coefficient are used to describe the interaction. Here the use of a simplified model, an affinity transport model, for determination of the affinity (K(D)) but not the kinetics (k(a), k(d)) has been investigated. When binding rates were highly governed by mass transport effects the two models returned the same affinity and gave similar residuals, but k(a) and k(d) values found with the kinetic transport model were unreliable. On the other hand the affinity transport model failed to describe the data when binding curves were less influenced by mass transport effects. Under such circumstances the kinetic transport model returned correct k(a) and k(d) values. Depending on the outcome of the analysis the affinity transport model can therefore be used to reduce uncertainties of the kinetic parameters or as an easy way to determine K(D) values from non-steady-state data. The use of the affinity transport model is illustrated with simulated data and with binding data obtained for the interaction between a 439 Da thrombin inhibitor and immobilized thrombin. Since it is more difficult to resolve high k(a) values for low molecular weight analytes, the affinity transport model may be particularly useful for affinity analysis involving fast reactions between such analytes and immobilized protein targets.     

Modulation of vitronectin receptor binding by membrane lipid composition.

The vitronectin (Vn) receptor belongs to the integrin family of proteins and although its biochemical structure is fully characterized little is known about its binding affinity and specificity. We report here that Vn receptor binding to different matrix proteins is influenced by the surrounding lipid composition of the membrane. Human placenta affinity purified Vn receptor was inserted into liposomes of different composition: (i) phosphatidylcholine (PC); (ii) PC+phosphatidylethanolamine (PE); (iii) PC+PE+phosphatidylserine (PS) + phosphatidylinositol (PI) + cholesterol (chol). The amount of purified material that could be incorporated into the three lipid vesicle preparations was proportional to the efficiency of the vesicle formation that increased from PC (38%) to PC+PE and PC+PE+PS+PI+chol (about 50%) vesicles. Electron microscopy analysis showed that the homogeneity and size of the three liposome preparations were comparable (20-nm diameter) but their binding capacity to a series of substrates differed widely. Vn receptor inserted in PC liposomes bound only Vn, but when it was inserted in PC+PE and PC+PE+PS+PI+chol liposomes it also attached to von Willebrand factor (vWF) and fibronectin (Fn). Vn receptor had higher binding capacity for substrates when it was inserted in PC+PE+PS+PI+chol than PC+PE liposomes. Antibodies to Vn receptor blocked Vn receptor liposome binding to Vn, vWF, and Fn. The intrinsic emission fluorescence spectrum of the Vn receptor reconstituted in PC+PE+PS+PI+chol liposomes was blue-shifted in relation to PC liposomes, suggesting a conformational change of the receptor in the membranes. These data provide direct evidence that the Vn receptor is "promiscuous" and can associate with Vn, vWF and Fn. The nature of the membrane lipid composition surrounding the receptor could thus influence its binding affinity, possibly by changing its conformation or exposure or both.     

Acyl chain-based molecular selectivity for HL60 cellular phosphatidylinositol and of phosphatidylcholine by phosphatidylinositol transfer protein alpha

Mammalian phosphatidylinositol transfer protein alpha (PITP) is an intracellular lipid transporter with a binding site that can accommodate a single molecule of phosphatidylinositol (PI) or phosphatidylcholine (PC). Phospholipids are a heterogeneous population of molecular species that can be distinguished by their characteristic headgroups as well as their acyl chains at the sn-1 and sn-2 position. In this study, we have defined the acyl chain preference for PITPalpha when presented with a total population of cellular lipids. Recombinant PITPalpha loaded with bacterial lipid, phosphatidylglycerol (PG), was incubated with permeabilised HL60 cells, followed by recovery of PITPalpha by affinity chromatography. Lipids extracted from the PITPalpha were analysed by tandem electrospray ionisation mass spectrometry (ESI-MS) and showed total exchange of acquired bacterial lipids for HL60 cellular PI and PC. Detailed comparison of the molecular species composition of bound phospholipids with those in whole cells permitted the assessment of selectivity of acyl chain binding. For both phospholipid classes, progressive fractional enrichments in bound species possessing shorter acyl chains were apparent with a preference order: 16:1>16:0>18:1>18:0>20:4. A recapitulation of this specificity order was also seen from a dramatically altered range of molecular species present in HL60 cells enriched with arachidonate over many weeks of culture. We speculate that short-chain, saturate-binding preferences under both conditions may reflect properties in vivo. This is consistent with target cell membranes actively remodelling newly delivered phospholipids after transport rather than relying on the transport of the specific molecular species conventionally found in mammalian membranes.     

Differential interfacial and substrate binding modes of mammalian pancreatic phospholipases A2: a comparison among human, bovine, and porcine enzymes.

To identify the residues essential for interfacial binding and substrate binding of human pancreatic phospholipase A2 (hpPLA2), several ionic residues in the putative interfacial binding surface (R6E, K7E, K10E, and K116E) and substrate binding site (D53K and K56E) were mutated. Interfacial affinity of these mutants was measured using anionic polymerized liposomes, and their enzymatic activity was measured using various substrates including phospholipid monomers, zwitterionic and anionic micelles, and anionic polymerized mixed liposomes. Similar mutations (R6E, K10E, K56E, and K116E) were made to porcine pancreatic phospholipase A2 (ppPLA2), and the properties of mutants were measured by the same methods. Results indicate that hpPLA2 and ppPLA2 have similar interfacial binding mechanisms in which cationic residues in the amino terminus and Lys-116 in the carboxy terminus are involved in binding to anionic lipid surfaces. Small but definite differences between the two enzymes were observed in overall interfacial affinity and activity and the effects of the mutations on interfacial enzyme activity. The interfacial binding of hpPLA2 and ppPLA2 is distinct from that of bovine pancreatic phospholipase A2 in that Lys-56 is involved in the interfacial binding of the latter enzyme. The unique phospholipid headgroup specificity of hpPLA2 derives from the presence of Asp-53 in the substrate binding site. This residue appears to participate in stabilizing electrostatic interactions with the cationic ethanolamine headgroup, hence the phosphatidylethanolamine preference of hpPLA2. Taken together, these studies reveal the similarities and the differences in the mechanisms by which mammalian pancreatic phospholipases A2 interact with lipid aggregates and perform interfacial catalysis.     

Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.

We studied the binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta) to vesicles containing the negatively charged phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). PLC-delta did not bind significantly to large unilamellar vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but bound strongly to vesicles formed from mixtures of PC and PIP2. The apparent association constant for the putative 1:1 complex formed between PLC-delta and PIP2 was Ka congruent to 10(5) M-1. The binding strength increased further (Ka congruent to 10(6) M-1) when the vesicles also contained 30% PS. High-affinity binding of PLC-delta to PIP2 did not require Ca2+. PLC-delta bound only weakly to vesicles formed from mixtures of PC and either PS or phosphatidylinositol (PI); binding increased as the mole fraction of acidic lipid in the vesicles increased. We also studied the membrane binding of a small basic peptide that corresponds to a conserved region of PLC. Like PLC-delta, the peptide bound weakly to vesicles containing monovalent negatively charged lipids; unlike PLC-delta, it did not bind strongly to vesicles containing PIP2. Our data suggest that a significant fraction of the PLC-delta in a cell could be bound to PIP2 on the cytoplasmic surface of the plasma membrane.     

The low-affinity lipid binding site of the non-specific lipid transfer protein. Implications for its mode of action.

The non-specific lipid transfer protein (nsL-TP) from bovine liver was studied by using the following fluorescent lipid analogs: phosphatidylcholine species with a sn-2-pyrenylacyl-chain of different length [Pyr(x)PC], sn-2-pyrenyldecanoyl-labelled phosphatidylinositol [Pyr(10)PI], -phosphatidylinositol 4-phosphate [Pyr(10)PIP], -phosphatidylinositol 4,5-bisphosphate [Pyr(10)PIP2] and dehydroergosterol. These analogs provided information on the effect of hydrophobicity and charge on lipid binding and transfer by nsL-TP. Binding of the Pyr(x)PC species decreased with increasing sn-2 acyl-chain length. Under equilibrium conditions, the fraction of nsL-TP that carried a PC molecule did not exceed 8%, which is consistent with a low affinity binding site. Also nsL-TP-mediated transfer of the Pyr(x)PC species decreased with increasing sn-2 acyl-chain length and was highly correlated with spontaneous transfer. Binding of the phosphoinositides increased in the order Pyr(10)PI less than Pyr(10)PIP less than Pyr(10)PIP2, indicating that an increase in lipid negative charge stimulates binding. The transfer of the phosphoinositides, however, decreased in the same order, which suggests that a high negative charge impairs the dissociation of the phospholipid from nsL-TP. Cholesterol, at concentrations up to 50 mol% in the donor membrane, hardly affected binding and transfer of Pyr(6)PC, strongly suggesting that nsL-TP has no high binding affinity for cholesterol. In agreement with this, binding of dehydroergosterol to nsL-TP was not detectable. Despite this apparently negligible affinity, nsL-TP-mediated transfer of dehydroergosterol was in the same order as that of Pyr(6)PC. The results are interpreted to indicate that transfer of lipids by nsL-TP involves the formation of a putative low-affinity lipid-protein complex. This formation is enhanced when lipid hydrophobicity decreases or lipid negative charge increases. Based on the binding and transfer data, the mode of action of nsL-TP is discussed in terms of change in free energy.     

Molecular mechanism of an oncogenic mutation that alters membrane targeting: Glu17Lys modifies the PIP lipid specificity of the AKT1 PH domain

The protein kinase AKT1 regulates multiple signaling pathways essential for cell function. Its N-terminal PH domain (AKT1 PH) binds the rare signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)], resulting in plasma membrane targeting and phosphoactivation of AKT1 by a membrane-bound kinase. Recently, it was discovered that the Glu17Lys mutation in the AKT1 PH domain is associated with multiple human cancers. This mutation constitutively targets the AKT1 PH domain to the plasma membrane by an unknown mechanism, thereby promoting constitutive AKT1 activation and oncogenesis. To elucidate the molecular mechanism underlying constitutive plasma membrane targeting, this work compares the membrane docking reactions of the isolated wild-type and E17K AKT1 PH domains. In vitro studies reveal that the E17K mutation dramatically increases the affinity for the constitutive plasma membrane lipid PI(4,5)P(2). The resulting PI(4,5)P(2) equilibrium affinity is indistinguishable from that of the standard PI(4,5)P(2) sensor, PLCdelta1 PH domain. Kinetic studies indicate that the effects of E17K on PIP lipid binding arise largely from electrostatic modulation of the dissociation rate. Membrane targeting analysis in live cells confirms that the constitutive targeting of E17K AKT1 PH to plasma membrane, like PLCdelta1 PH, stems from PI(4,5)P(2) binding. Overall, the evidence indicates that the molecular mechanism underlying E17K oncogenesis is a broadened target lipid selectivity that allows high-affinity binding to PI(4,5)P(2). Moreover, the findings strongly implicate the native Glu17 side chain as a key element of PIP lipid specificity in the wild-type AKT1 PH domain. Other PH domains may employ an analogous anionic residue to control PIP specificity.     

Introduction of mannose binding protein-type phosphatidylinositol recognition into pulmonary surfactant protein A.

Pulmonary surfactant protein A (SP-A) and mannose-binding protein A (MBP-A) are collectins in the C-type lectin superfamily. These collectins exhibit unique lipid binding properties. SP-A binds to dipalmitoyl phosphatidylcholine (DPPC) and galactosylceramide (GalCer) and MBP-A binds to phosphatidylinositol (PI). SP-A also interacts with alveolar type II cells. Monoclonal antibodies (mAbs PE10 and PC6) that recognize human SP-A inhibit the interactions of SP-A with lipids and alveolar type II cells. We mapped the epitopes for anti-human SP-A mAbs by a phage display peptide library. Phage selected by mAbs displayed the consensus peptide sequences that are nearly identical to 184TPVNYTNWYRG194 of human SP-A. The synthetic peptide GTPVNYTNWYRG completely blocked the binding of mAbs to human SP-A. Chimeric proteins were generated in which the rat SP-A region Thr174-Gly194 or the human SP-A region Ser174-Gly194 was replaced with the MBP-A region Thr164-Asp184 (rat ama4 or hu ama4, respectively). The mAbs failed to bind hu ama4. Rat ama4 bound to an affinity matrix on mannose-sepharose but lost all of the SP-A functions except carbohydrate binding and Ca2+-independent GalCer binding. Strikingly, the rat ama4 chimera acquired the PI binding property that MBP-A exhibits. This study demonstrates that the amino acid residues 174-194 of SP-A and the corresponding region of MBP-A are critical for SP-A-type II cell interaction and Ca2+-dependent lipid binding of collectins.