Structure and dynamics of the phospholipase C-delta1 pleckstrin homology domain located at the lipid bilayer surface

Despite the importance of signal transduction pathways at membrane surfaces, there have been few means of investigating their molecular mechanisms based on the structural information of membrane-bound proteins. We applied solid state NMR as a novel method to obtain structural information about the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain at the lipid bilayer surface. NMR spectra of the alanine residues in the vicinity of the beta5/beta6 loop in the PH domain revealed changes in local conformations due to the membrane localization of the protein. We propose that these conformational changes originate from a hydrophobic interaction between the amphipathic alpha-helix located in the beta5/beta6 loop and the hydrophobic layer of the membrane and contribute to the membrane binding affinity, interdomain interactions and intermolecular interactions of PLC-delta1.     

Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C-delta1

The membrane binding affinity of the pleckstrin homology (PH) domain of phospholipase C (PLC)-delta1 was investigated using a vesicle coprecipitation assay and the structure of the membrane-associated PH domain was probed using solid-state (13)C NMR spectroscopy. Twenty per cent phosphatidylserine (PS) in the membrane caused a moderate but significant reduction of the membrane binding affinity of the PH domain despite the predicted electrostatic attraction between the PH domain and the head groups of PS. Solid-state NMR spectra of the PH domain bound to the phosphatidylcholine (PC)/PS/phosphatidylinositol 4,5-bisphosphate (PIP(2)) (75 : 20 : 5) vesicle indicated loss of the interaction between the amphipathic alpha2-helix of the PH domain and the interface region of the membrane which was previously reported for the PH domain bound to PC/PIP(2) (95 : 5) vesicles. Characteristic local conformations in the vicinity of Ala88 and Ala112 induced by the hydrophobic interaction between the alpha2-helix and the membrane interface were lost in the structure of the PH domain at the surface of the PC/PS/PIP(2) vesicle, and consequently the structure becomes identical to the solution structure of the PH domain bound to d-myo-inositol 1,4,5-trisphosphate. These local structural changes reduce the membrane binding affinity of the PH domain. The effects of PS on the PH domain were reversed by NaCl and MgCl(2), suggesting that the effects are caused by electrostatic interaction between the protein and PS. These results generally suggest that the structure and function relationships among PLCs and other peripheral membrane proteins that have similar PH domains would be affected by the local lipid composition of membranes.     

Activity of beta3-beta4 loop of the PH domain is required for the membrane targeting of SWAP-70

SWAP-70 translocates to the plasma membrane in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner and contributes to membrane ruffling. It binds to phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) through its PH domain, which is essential for the membrane translocation after EGF stimulation. We examined the behavior of the SWAP-70s which have mutations in the beta3/beta4 loop of the PH domain. The two mutants fused to green fluorescent protein (GFP) carrying the mutations failed to translocate to the plasma membrane. The sole PH domains carrying the same mutations behaved similarly. The PtdIns(3,4,5)P(3) binding activity of two mutants was comparable to that of the wild-type protein. These results suggest that translocation of SWAP-70 largely depends on the activity of the PH domain, and that not only PtdIns(3,4,5)P(3) binding activity, but also some additional activity of the PH domain is required for the translocation.     

Direct observation of alpha-lactalbumin,adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures

The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (α-La). α-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize α-La-membrane mechanisms of interaction and α-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imaging, we are able to distinguish between protein adsorption and insertion in the membranes. Our results indicate that, upon addition of α-La to giant vesicles samples, protein is inserted into the lipid bilayer with rates that are concentration-dependent. The formation of heterogeneous hybrid protein-lipid co-aggregates, paralleled with protein conformational and structural changes, alters the membrane structure and morphology, leading to an increase in membrane fluidity.     

Conformation of membrane-associated proapoptotic tBid

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.     

SWAP-70 associates transiently with macropinosomes

Cells accomplish the non-selective uptake of extracellular fluids, antigens and pathogens by the endocytic process of macropinocytosis. The protein SWAP-70 is a widely expressed, pleckstrin-homology (PH) domain-containing protein that marks a transitional subset of actin filaments in motile cells. Here we report that the protein SWAP-70 associates transiently with macropinosomes in dendritic cells and NIH/3T3 fibroblasts. The association of SWAP-70 with macropinosomes is preceded by the accumulation of Rac-GTP and followed by that of Rab5. Three regions of SWAP-70, the N-terminal region, the PH domain and the C-terminal region, contribute in a combinatorial manner to the transient association with newly formed macropinosomes in the cell periphery and occasionally with aged macropinosomes on their passage to the cell center. These data identify SWAP-70 as a transient component of early macropinosomes.     

The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles in toxin oligomerization and pore formation

The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a β-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the β-sheet, allowing insertion of β-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of β-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study

The second messenger lipid PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3)-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP(3)-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3) lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3). The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP(3) target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP(3) on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3) headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3) headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3) headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP(3)-bound GRP1 PH domain on supported lipid bilayers.     

Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins

The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of MARCKS reversibly sequesters a significant fraction of the L-alpha-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) on the plasma membrane. To test this, we utilize three techniques that measure the ability of a peptide corresponding to its effector domain, MARCKS(151-175), to sequester PIP2 in model membranes containing physiologically relevant fractions (15-30%) of the monovalent acidic lipid phosphatidylserine. First, we measure fluorescence resonance energy transfer from Bodipy-TMR-PIP2 to Texas Red MARCKS(151-175) adsorbed to large unilamellar vesicles. Second, we detect quenching of Bodipy-TMR-PIP2 in large unilamellar vesicles when unlabeled MARCKS(151-175) binds to vesicles. Third, we identify line broadening in the electron paramagnetic resonance spectra of spin-labeled PIP2 as unlabeled MARCKS(151-175) adsorbs to vesicles. Theoretical calculations (applying the Poisson-Boltzmann relation to atomic models of the peptide and bilayer) and experimental results (fluorescence resonance energy transfer and quenching at different salt concentrations) suggest that nonspecific electrostatic interactions produce this sequestration. Finally, we show that the PLC-delta1-catalyzed hydrolysis of PIP2, but not binding of its PH domain to PIP2, decreases markedly as MARCKS(151-175) sequesters most of the PIP2.     

Acidic interaction of the colicin A pore-forming domain with model membranes of Escherichia coli lipids results in a large perturbation of acyl chain order and stabilization of the bilayer.

2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs.     

Fluorescence analysis of the lipid binding-induced conformational change of apolipoprotein e4

Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipid-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL(3) suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL(3).     

Membrane interactions of the sodium channel S4 segment and its fluorescently-labeled analogues.

A 24-amino acid peptide corresponding to the S4 segment of the sodium channel was synthesized. In order to perform fluorescence energy transfer measurements and to monitor the interaction of the peptide with lipid vesicles, the peptide was selectively labeled with fluorescence probes at either its N- or C-terminal amino acids. The fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazol-4- yl-(NBD-)labeled analogues displayed blue shifts upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. The results revealed that both the N- and C-terminus of the S4 segment are located within the lipid bilayer. Titration of solutions containing NBD-labeled peptides with SUV was used to generate binding isotherms, from which surface partition constants, in the range of 10(4) M-1, were derived. The shape of the binding isotherms as well as fluorescence energy transfer measurements suggest that aggregation of peptide monomers within the membrane readily occurs in acidic but not in zwitterionic vesicles. Furthermore, the results provide good correlation between the incidence of aggregation in PC/PS vesicles and the ability of the peptides to permeate the vesicle's membrane. However, a transmembrane diffusion potential had no detectable effect on the location of the peptide within the lipid bilayer or on its aggregation state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31-32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively (13)C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged (1)H-(13)C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain (2)H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.     

Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin

Dynamin, a large GTPase, is located at the necks of clathrin-coated pits where it facilitates the release of coated vesicles from the plasma membrane upon GTP binding, and hydrolysis. Previously, we have shown by negative stain electron microscopy that wild-type dynamin and a dynamin mutant lacking the C-terminal proline-rich domain, DeltaPRD, form protein-lipid tubes that constrict and vesiculate upon addition of GTP. Here, we show by time-resolved cryo-electron microscopy (cryo-EM) that DeltaPRD dynamin in the presence of GTP rapidly constricts the underlying lipid bilayer, and then gradually disassembles from the lipid. In agreement with the negative stain results, the dynamin tubes constrict from 50 to 40 nm, and their helical pitch decreases from approximately 13 to 9.4 nm. However, in contrast to the previous results, examination by cryo-EM shows that the lipid bilayer remains intact and small vesicles or fragments do not form upon GTP binding and hydrolysis. Therefore, the vesicle formation seen by negative stain may be due to the lack of mobility of the dynamin tubes on the grid during the GTP-induced conformational changes. Our results confirm that dynamin is a mechanochemical enzyme and suggest that during endocytosis dynamin is directly responsible for membrane constriction. In the cell, other proteins may enhance the activity of dynamin or the constraints induced by the surrounding coated pit and plasma membrane during constriction may cause the final membrane fission event.     

Synaptotagmin’s Role in Neurotransmitter Release Likely Involves Ca-induced Conformational Transition

Neuronal exocytosis is mediated by a Ca²⁺-triggered membrane fusion event that joins synaptic vesicles and presynaptic membrane. In this event, synaptotagmin I plays a key role as a Ca²⁺ sensor protein that binds to and bends the presynaptic membrane with its C2B domain, and thereby initiates membrane fusion. We report free energy calculations according to which C2B-induced membrane bending is preceded by a Ca²⁺- and membrane-dependent conformational transition. In this transition C2B attaches to the membrane, moves its C-terminal helix from the orientation seen in the available (but membrane-free) crystal/NMR structures as pointing away from the membrane (helix-up), to an orientation pointing toward the membrane (helix-down). In the C2B helix-down state, lipid tails in the proximal membrane bilayer leaflet interact with the moved helix and become disordered, whereas tails in the distal leaflet, to keep in contact with the proximal leaflet, become stretched and ordered. The difference in lipid tail packing between the two leaflets results in an imbalance of pressure across the membrane, and thereby causes membrane bending. The lipid-disordering monitored in the simulations is well suited to facilitate Ca²⁺-triggered membrane fusion.     

Synaptotagmin’s Role in Neurotransmitter Release Likely Involves Ca2+-induced Conformational Transition

Neuronal exocytosis is mediated by a Ca²⁺-triggered membrane fusion event that joins synaptic vesicles and presynaptic membrane. In this event, synaptotagmin I plays a key role as a Ca²⁺ sensor protein that binds to and bends the presynaptic membrane with its C2B domain, and thereby initiates membrane fusion. We report free energy calculations according to which C2B-induced membrane bending is preceded by a Ca²⁺- and membrane-dependent conformational transition. In this transition C2B attaches to the membrane, moves its C-terminal helix from the orientation seen in the available (but membrane-free) crystal/NMR structures as pointing away from the membrane (helix-up), to an orientation pointing toward the membrane (helix-down). In the C2B helix-down state, lipid tails in the proximal membrane bilayer leaflet interact with the moved helix and become disordered, whereas tails in the distal leaflet, to keep in contact with the proximal leaflet, become stretched and ordered. The difference in lipid tail packing between the two leaflets results in an imbalance of pressure across the membrane, and thereby causes membrane bending. The lipid-disordering monitored in the simulations is well suited to facilitate Ca²⁺-triggered membrane fusion.     

Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

'In dopaminergic neurons, α-synuclein (αS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of αS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free state and the lipid-bound αS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s^− 1. Partitioning of the binding modes is dependent on lipid/αS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of αS adopts an α-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this “dark state.” Titration of αS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by αS.     

A fluorescence and CD study on the interaction of synthetic lipophilic hepatitis B virus preS(120–145) peptide analogues with phospholipid vesicles

The interaction of the immunogenic peptide of human hepatitis B virus (HBV) preS(120–145), including B and T epitopes, with phospholipid vesicles has been studied by fluorescence techniques and CD. In addition, interaction of three lipopeptides derived from preS(120–145) containing stearoyl, cholanoyl, and tripalmitoyl-S-glyceryl-cysteine (Pam₃C) SS moieties with dipalmitoylphosphatidylcholine (DPPC) has been investigated by polarization fluorescence spectroscopy. Fluorescence experiments showed an increase in fluorescence intensity and a blue shift of the maximum emission wavelength upon interaction of preS(120–145) with DPPC vesicles below the transition temperature (T_c), indicating that the tryptophan moiety enters a more hydrophobic environment. Moreover, fluorescence polarization experiments showed that the peptide decreased the membrane fluidity at the hydrophobic core, increasing the T_c of the lipid and decreasing the amplitude of the change of fluorescence polarization associated with the cooperative melting of 1,6-diphenyl-1,3,5-hexatriene labeled vesicles. The absence of leakage of vesicle-entrapped carboxyfluorescein indicates that the peptide did not promote vesicle lysis. Besides, the three lipopeptides derived from preS(120–145) showed a more pronounced rigidifying effect at the hydrophobic core of the bilayer, with a significative increase in the T_c. Stearoyl- and cholanoyl-preS(120–145) restricted the motion of lipids also at the polar surface, whereas Pam₃CSS-preS(120–145) did not alter the polar head group order. Finally, CD studies in 2,2,2-triflouroethanol or in presence of vesicles suggested that the bound peptide adopted amphiphilic α-helical and β-sheet structures, with an important contribution of the β-turn. It is concluded that preS(120–145) can interact with the lipid membrane through the formation of an amphipathic structure combination of β-sheet and α-helix aligned parallel to the membrane surface, involving the N-terminal residues, and penetrating only a short distance into the hydrophobic core. The C-terminal part, with a combination of β-turn and β-sheet structure, remains at the outer part of the bilayer, being potentially accessible to immunocompetent cells. Furthermore, coupling of an hydrophobic moiety to the N-terminal part of the peptide favors anchoring to the membrane, probably facilitating interaction of the peptide with the immunoglobulin receptor. These results are in agreement with the induction of immune response by preS(120–145) and with the enhanced immunogenicity found in general for lipid-conjugated immunopeptides. © 1996 John Wiley & Sons, Inc.