Evidence for lipid packaging in the crystal structure of the GM2-activator complex with platelet activating factor

GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2A crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement of two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals' contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.     

Structural analysis of lipid complexes of GM2-activator protein

The GM2-activator protein (GM2-AP) is a small lysosomal lipid transfer protein essential for the hydrolytic conversion of ganglioside GM2 to GM3 by beta-hexosaminidase A. The crystal structure of human apo-GM2-AP is known to consist of a novel beta-cup fold with a spacious hydrophobic interior. Here, we present two new structures of GM2-AP with bound lipids, showing two different lipid-binding modes within the apolar pocket. The 1.9A structure with GM2 bound shows the position of the ceramide tail and significant conformational differences among the three molecular copies in the asymmetric unit. The tetrasaccharide head group is not visible and is presumed to be disordered. However, its general position could be established through modeling. The structure of a low-pH crystal, determined at 2.5A resolution, has a significantly enlarged hydrophobic channel that merges with the apolar pocket. Electron density inside the pocket and channel suggests the presence of a trapped phospholipid molecule. Structure alignments among the four crystallographically unique monomers provide information on the potential role for lipid binding of flexible chain segments at the rim of the cavity opening. Two discrete orientations of the S130-T133 loop define an open and a closed configuration of the hydrophobic channel that merges with the apolar pocket. We propose: (i) that the low-pH structure represents an active membrane-binding conformation; (ii) that the mobile S130-T133 loop serves as a gate for passage of ligand into the apolar pocket; and (iii) that this loop and the adjacent apolar V59-W63 loop form a surface patch with two exposed tryptophan residues that could interface with lipid bilayers.     

Import of lyso-phosphatidylcholine into chloroplasts likely at the origin of eukaryotic plastidial lipids

Plastids rely on the import of extraplastidial precursor for the synthesis of their own lipids. This key phenomenon in the formation of plastidial phosphatidylcholine (PC) and of the most abundant lipids on earth, namely galactolipids, is poorly understood. Various suggestions have been made on the nature of the precursor molecule(s) transferred to plastids, but despite general agreement that PC or a close metabolite plays a central role, there is no clear-cut answer to this question because of a lack of conclusive experimental data. We therefore designed experiments to discriminate between a transfer of PC, 1-acylglycero phosphorylcholine (lyso-PC), or glycerophosphorylcholine. After pulse-chase experiments with glycerol and acetate, plastids of leek (Allium porrum L.) seedlings were purified. The labels of the glycerol moiety and the sn-1- and sn-2-bound fatty acids of plastidial lipids were determined and compared with those associated with the extraplastidial PC. After import, plastid lipids contained the glycerol moiety and the fatty acids esterified to the sn-1 position originating from the extraplastidial PC; no import of sn-2-bound fatty acid was detected. These results rule out a transfer of PC or glycerophosphorylcholine, and are totally explained by an import of lyso-PC molecules used subsequently as precursor for the synthesis of eukaryotic plastid lipids.     

Crystal Structures of Cholesteryl Ester Transfer Protein in Complex with Inhibitors

Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles.     

Interactions of the GM2 Activator Protein with Phosphatidylcholine Bilayers: A Site-Directed Spin-Labeling Power Saturation Study

The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.     

Lipid-Free Antigen B Subunits from Echinococcus granulosus: Oligomerization,Ligand Binding,and Membrane Interaction Properties

BackgroundThe hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.Conclusions/SignificanceWe show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.     

The role of sphingolipid metabolism in cutaneous permeabilitybarrier formation

The epidermal permeability barrier of mammalian skin is localized in the stratum corneum. Corneocytes are embedded in an extracellular, highly ordered lipid matrix of hydrophobic lipids consisting of about 50% ceramides, 25% cholesterol and 15% long and very long chain fatty acids. The most important lipids for the epidermal barrier are ceramides. The scaffold of the lipid matrix is built of acylceramides, containing ω-hydroxylated very long chain fatty acids, acylated at the ω-position with linoleic acid. After glucosylation of the acylceramides at Golgi membranes and secretion, the linoleic acid residues are replaced by glutamate residues originating from proteins exposed on the surface of corneocytes. Removal of their glucosyl residues generates a hydrophobic surface on the corneocytes used as a template for the formation of extracellular lipid layers of the water permeability barrier. Misregulation or defects in the formation of extracellular ceramide structures disturb barrier function. Important anabolic steps are the synthesis of ultra long chain fatty acids, their ω-hydroxylation, and formation of ultra long chain ceramides and glucosylceramides. The main probarrier precursor lipids, glucosylceramides and sphingomyelins, are packed in lamellar bodies together with hydrolytic enzymes such as glucosylceramide-β-glucosidase and acid sphingomyelinase and secreted into the intercelullar space between the stratum corneum and stratum granulosum. Inherited defects in the extracellular hydrolytic processing of the probarrier acylglucosylceramides impair epidermal barrier formation and cause fatal diseases: such as prosaposin deficiency resulting in lack of lysosomal lipid binding and transfer proteins, or the symptomatic clinical picture of the “collodion baby” in the absence of glucocerebrosidase. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

PI transfer protein: the specific recognition of phospholipids and its functions

Phosphatidylinositol transfer proteins (PITPs) can bind specifically and transfer a single phosphatidylinositol (PI) molecule between phospholipid membranes in an ATP-independent manner in vitro. PITPs exist in all the eukaryotic systems from yeast to human. PITP plays an essential role in intracellular vesicle flow and inositol lipid signaling. The crystal structure of yeast PITP Sec14p reveals a large hydrophobic pocket to accommodate the acyl chains of phospholipid molecules. At the opening of the pocket, a hydrogen bond network may render Sec14p the binding specificity to PI molecules. The structure suggests that the PI-binding ability may play an important role in the in vivo function of PITPs.     

Modification of the fatty acid composition of individual phospholipids and neutral lipids after infection of the simian erythrocyte by Plasmodium knowlesi

Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlesi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate (+23%), oleate (+29%) and linoleate (+89%), but contained less stearate (-27%) and arachidonate (-40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (-60%), but their level is increased in PI.     

Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity

To better understand the mechanism of lipid transfer protein (LTP) action and the effects of altered lipoprotein composition on its activity, we evaluated the dependence of LTP activity on the concentrations of cholesteryl ester (CE) and/or triglyceride (TG) in the phospholipid bilayer of substrate particles. Phosphatidylcholine (PC)-cholesterol liposomes containing up to 2 mole% TG and/or CE were prepared by cholate dialysis and used as either the donor of lipids to, or the acceptor of lipids from, low density lipoproteins (LDL). CE or TG transfer from liposomes of varying neutral lipid content to LDL showed saturation kinetics with an apparent Km of less than or equal to 0.2 mole%. Throughout this concentration-dependent response. PC transfer, which depended on the same LTP-donor particle binding interactions as those required for neutral lipid transfer, was essentially unchanged. Lipid transfer in the reverse direction (from LDL to liposomes of varying neutral lipid content) followed the same kinetics showing that transfer between the two particles is tightly coupled and bidirectional. When liposomes contained both TG and CE, these lipids competed for transfer in a manner analogous to that previously noted with lipoprotein substrates. In conclusion, CE and TG transfer activities are determined by the concentration of these lipids in the phospholipid surface of donor and acceptor particles. At low TG and CE concentrations, LTP bound to the liposome surface as indicated by PC transfer, but only a portion of these interactions actually facilitated a neutral lipid transfer event. Thus, the overall rate of neutral lipid transfer, and the competition between TG and CE for transfer, depend on the concentrations of these lipids in the phospholipid layer.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Membrane‐binding mechanism of a bacterial phospholipid N‐methyltransferase

The membrane lipid phosphatidylcholine (PC) is crucial for stress adaptation and virulence of the plant pathogen Agrobacterium tumefaciens. The phospholipid N‐methyltransferase PmtA catalyzes three successive methylations of phosphatidylethanolamine to yield PC. Here, we asked how PmtA is recruited to its site of action, the inner leaflet of the membrane. We found that the enzyme attaches to the membrane via electrostatic interactions with anionic lipids, which do not serve as substrate for PmtA. Increasing PC concentrations trigger membrane dissociation suggesting that membrane binding of PmtA is negatively regulated by its end product PC. Two predicted alpha‐helical regions (αA and αF) contribute to membrane binding of PmtA. The N‐terminal helix αA binds anionic lipids in vitro with higher affinity than the central helix αF. The latter undergoes a structural transition from disordered to α‐helical conformation in the presence of anionic lipids. The basic amino acids R8 and K12 and the hydrophobic amino acid F19 are critical for membrane binding by αA as well as for activity of full‐length PmtA. We conclude that a combination of electrostatic and hydrophobic forces is responsible for membrane association of the phospholipid‐modifying enzyme.     

Structure of sterol carrier protein 2 at 1.8 A resolution reveals a hydrophobic tunnel suitable for lipid binding

Sterol carrier protein 2, also known as nonspecific lipid transfer protein is a ubiquitous, small, basic protein of 13 kDa found in animals. Its primary structure is highly conserved between different species, and it has been implicated in the intracellular transport of lipids and in a wide range of other in vitro functions related to sterol and fatty acid metabolism. Sterol carrier protein 2 deficiency in mice leads to elevated concentrations of phytanic acid in the serum and causes hepatocarcinogenesis. However, its actual physiological role is still unknown. Although sterol carrier protein 2 has been studied extensively in the past 20 years, very little is known concerning its three-dimensional structure. The crystal structure of rabbit sterol carrier protein 2, determined at 1.8 A resolution with the MIRAS method, shows a unique alpha/beta-fold. The core of this protein forms a five-stranded antiparallel beta-sheet flanked by five helices. A C-terminal segment (residues 114-123), together with part of the beta-sheet and four alpha-helices, form a hydrophobic tunnel providing the environment for apolar ligands such as fatty acids and fatty acyl-coenzyme As. Structurally well-characterized nonspecific lipid transfer proteins from plants have hydrophobic tunnel-like cavities, which were identified as the binding site for fatty acids and related apolar ligands. Despite the fact that plant nonspecific lipid transfer proteins are smaller proteins than sterol carrier protein 2, show no sequence homology to sterol carrier protein 2, and are structurally unrelated, the cavities of these two classes of proteins are very similar with respect to size, shape, and hydrophobicity, suggesting a common functional role.     

Lipid binding in rice nonspecific lipid transfer protein-1 complexes from Oryza sativa

Nonspecific lipid transfer proteins (nsLTPs) facilitate the transfer of phospholipids, glycolipids, fatty acids and steroids between membranes, with wide-ranging binding affinities. Three crystal structures of rice nsLTP1 from Oryza sativa, complexed with myristic (MYR), palmitic (PAL) or stearic acid (STE) were determined. The overall structures of the rice nsLTP1 complexes belong to the four-helix bundle folding with a long C-terminal loop. The nsLTP1-MYR and the nsLTP1-STE complexes bind a single fatty acid while the nsLTP1-PAL complex binds two molecules of fatty acids. The C-terminal loop region is elastic in order to accommodate a diverse range of lipid molecules. The lipid molecules interact with the nsLTP1-binding cavity mainly with hydrophobic interactions. Significant conformational changes were observed in the binding cavity and the C-terminal loop of the rice nsLTP1 upon lipid binding.     

Synthesis of novel NBD-GM1 and NBD-GM2 for the transfer activity of GM2-activator protein by a FRET-based assay system

The ganglioside-activator protein is an essential cofactor for the lysosomal degradation of ganglioside GM2 (GM2) by beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interphase. Mutations in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. In order to efficiently and sensitively probe the glycolipid binding and membrane activity of this cofactor, we synthesized two new fluorescent glycosphingolipid (GSL) probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multistep synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed us to introduce the chromophore into the region between the polar head group and the hydrophobic anchor of the lipid. Both fluorescent glycolipids exhibited an extremely low off-rate in model membranes and displayed very efficient resonance energy transfer to rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE) as acceptor. The binding to GM2-activator protein (GM2AP) and the degrading enzyme was shown to be unaltered compared to their natural analogues. A novel fluorescence-resonance energy transfer (FRET) assay was developed to monitor in real time the protein-mediated intervesicular transfer of these lipids from donor to acceptor liposomes. The data obtained indicate that this rapid and robust system presented here should serve as a valuable tool to probe quantitatively and comprehensively the membrane activity of GM2AP and other sphingolipid activator proteins and facilitate further structure-function studies aimed at delineating independently the lipid- and the enzyme-binding mode of these essential cofactors.     

Ligand Extraction Properties of the GM2 Activator Protein and Its Interactions with Lipid Vesicles

The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.     

A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) belong to the vitellogenin (VTG) family of lipid transfer proteins. MTP is essential for the intracellular assembly and secretion of apoB-containing lipoproteins, the key intravascular lipid transport proteins in vertebrates. We report the predicted three-dimensional structure of the C-terminal lipid binding cavity of MTP, modeled on the crystal structure of the lamprey VTG gene product, lipovitellin. The cavity in MTP resembles those found in the intracellular lipid-binding proteins and bactericidal/permeability-increasing protein. Two conserved helices, designated A and B, at the entrance to the MTP cavity mediate lipid acquisition and binding. Helix A (amino acids 725-736) interacts with membranes in a manner similar to viral fusion peptides. Mutation of helix A blocks the interaction of MTP with phospholipid vesicles containing triglyceride and impairs triglyceride binding. Mutations of helix B (amino acids 781-786) and of N780Y, which causes abetalipoproteinemia, have no impact on the interaction of MTP with phospholipid vesicles but impair triglyceride binding. We propose that insertion of helix A into lipid membranes is necessary for the acquisition of neutral lipids and that helix B is required for their transfer to the lipid binding cavity of MTP.     

Tissue-specific Functions in the Fatty Acid-binding Protein Family

The intracellular fatty acid-binding proteins (FABPs) are abundantly expressed in almost all tissues. They exhibit high affinity binding of a single long-chain fatty acid, with the exception of liver FABP, which binds two fatty acids or other hydrophobic molecules. FABPs have highly similar tertiary structures consisting of a 10-stranded antiparallel β-barrel and an N-terminal helix-turn-helix motif. Research emerging in the last decade has suggested that FABPs have tissue-specific functions that reflect tissue-specific aspects of lipid and fatty acid metabolism. Proposed roles for FABPs include assimilation of dietary lipids in the intestine, targeting of liver lipids to catabolic and anabolic pathways, regulation of lipid storage and lipid-mediated gene expression in adipose tissue and macrophages, fatty acid targeting to β-oxidation pathways in muscle, and maintenance of phospholipid membranes in neural tissues. The regulation of these diverse processes is accompanied by the expression of different and sometimes multiple FABPs in these tissues and may be driven by protein-protein and protein-membrane interactions.     

Specificity of the phosphatidylcholine exchange protein from bovine liver

The phosphatidylcholine exchange protein from bovine liver stimulates the specific transfer of phosphatidylcholine (PC) from rat liver microsomes to mitochondria or phospholipid vesicles (Wirtz, K.W.A., Kamp, H.H., and van Deenen, L.L.M. (1972), Biochim. Biophys. Acta 274, 606). In the present study, it has been established which components of the PC molecule are essential to the specific interaction with the protein. Radiochemically labeled analogues of PC have been synthesized with modifications in the polar and apolar moiety, and their transfer was measured between donor and acceptor vesicles. Relative to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (egg yolk PC), transfer is inhibited or abolished when (a) the distance between phosphorus and nitrogen is decreased or increased and (b) a methyl group on the quaternary nitrogen is removed or substituted by an ethyl or propyl group. Transfer is much less affected when (a) the ester bonds are replaced by ether or carbon-carbon bonds, (b) the PC molecule contains two saturated fatty acids, and (c) the D stereoisomer is used. It is concluded that the protein has a binding site which interacts specifically with the phosphorylcholine head group and which cannot accommodate substantial configurational changes. Interaction with the apolar moiety of PC is less specific. However, lyso-PC is not transferred, suggesting that two hydrocarbon chains are required to stabilize the exchange protein-phospholipid complex. Interaction of [14C]PC-labeled exchange protein with vesicles of different phospholipid compositon has been analyzed by measuring the release of [14C]PC into these vesicles. Vesicles of egg PC or dimethylphosphatidylethanolamine function as acceptors, in contrast to vesicles of sphingomyelin or phosphatidylethanolamine.     

Structural and functional determinants of human plasma phospholipid transfer protein activity as revealed by site-directed mutagenesis of charged amino acids

Human plasma phospholipid transfer protein (PLTP) exchanges phospholipids between lipoproteins and remodels high-density lipoproteins (HDLs). We determined phospholipid transfer activity and HDL binding ability in wild-type PLTP and in 16 PLTP variants created by replacing 12 charged amino acids by site-directed mutagenesis. The data were analyzed in relation to the structure of a member of the same gene family, bactericidal/permeability-increasing protein, which is a boomerang-shaped molecule containing two symmetrical, hydrophobic pockets that bind phospholipid molecules. When expressed in COS-7 cells, wild-type and all mutant PLTPs accumulated intracellularly to nearly the same extent. Relative to wild-type PLTP, substitution(s) for amino acids with a lateral position totally exposed to the solvent produced reductions in transfer activity proportional to the reductions in the level of HDL binding. Variants containing substitutions for charged amino acids on the concave surface of PLTP did not affect binding to HDL or specific transfer activity. A mutation in the C-terminal pocket (E270R) led to a decrease in both the specific transfer activity and the level of binding to HDLs, whereas mutations in the N-terminal pocket (R25E and D231R) resulted in a large decrease in specific transfer activity without affecting HDL binding. The data support a model of transfer in which N- and C-terminal pockets have different roles in HDL binding and transfer activity. The N-terminal pocket may be critical to PLTP transfer activity but may have no involvement in binding to lipoproteins, whereas amino acid substitutions in the C-terminal pocket might reduce PLTP activity by decreasing PLTP's affinity for HDLs.