Contribution of each membrane binding domain of the CTP:phosphocholine cytidylyltransferase-alpha dimer to its activation, membrane binding, and membrane cross-bridging

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.     

Lipid-induced conformational switch in the membrane binding domain of CTP:phosphocholine cytidylyltransferase: a circular dichroism study

CTP:phosphocholine cytidylyltranferase (CCT) regulates phosphatidylcholine (PC) biosynthesis. Its activity is controlled by reversible interactions with membrane lipids, mediated by an internal segment referred to as domain M. Although domain M peptides adopt an amphipathic alpha-helical structure when membrane bound, the structure of this domain in the context of the whole enzyme in the lipid-free and lipid-bound state is unknown. Here we derive lipid-induced secondary structural changes in CCTalpha using circular dichroism and three deconvolution programs. The analysis of two fragments, CCT236 (CCT1-236, housing the catalytic domain) and a synthetic domain M peptide (CCT237-293) aided the assignment of structural change to specific domains. To carry out this study, we developed a micellar lipid activating system that would avoid generation of CCT-induced lipid vesicle aggregates that interfere with the CD analysis. Lysophosphatidylcholine/phosphatidylglycerol (LPC/PG) mixed micelles supported full activation of CCT and caused an increase in the alpha-helix content of full-length CCT from 25 to 41%, at the expense of all other conformations. LPC/PG also induced an increase in alpha-helix content of the domain M peptide from 7 to 85% at the expense of all other conformers. This lipid system did not significantly affect the secondary structure of CCT236, nor did it affect the proteolytic fragmentation pattern of this region within full-length CCT, suggesting that the region containing the catalytic domain changes very little upon membrane activation of CCT. Our data suggest that lipids trigger a conformational switch in domain M from a mixed structure to an alpha-helix, thus creating a hydrophobic face for membrane insertion. Our results negate the idea that domain M is entirely helical in both the soluble and membrane-bound forms of CCT.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with ²H- and ³¹P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.     

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB_1-23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB_1-23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.     

Activity and characterization of a pH-sensitive antimicrobial peptide

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. Previous work showed that when Histidine was incorporated into the peptide C18G it lost antimicrobial activity. The role of pH on activity and biophysical properties of the peptide was investigated to explain this phenomenon. Minimal inhibitory concentration (MIC) results demonstrated that decreased media pH increased antimicrobial activity. Trichloroethanol (TCE) quenching and red-edge excitation spectroscopy (REES) showed a clear pH dependence on peptide aggregation in solution. Trp fluorescence was used to monitor binding to lipid vesicles and demonstrated the peptide binds to anionic bilayers at all pH values tested, however, binding to zwitterionic bilayers was enhanced at pH 7 and 8 (above the His pKa). Dual Quencher Analysis (DQA) confirmed the peptide inserted more deeply in PC:PG and PE:PG membranes, but could insert into PC bilayers at pH conditions above the His pKa. Bacterial membrane permeabilization assays which showed enhanced membrane permeabilization at pH 5 and 6 but vesicle leakage assays indicate enhanced permeabilization of PC and PC:PG bilayers at neutral pH. The results indicate the ionization of the His side chain affects the aggregation state of the peptide in solution and the conformation the peptide adopts when bound to bilayers, but there are likely more subtle influences of lipid composition and properties that impact the ability of the peptide to form pores in membranes.     

Effect of lipid composition on buforin II structure and membrane entry

Buforin II is a 21-amino acid polycationic antimicrobial peptide derived from a peptide originally isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. It is hypothesized to target a wide range of bacteria by translocating into cells without membrane permeabilization and binding to nucleic acids. Previous research found that the structure and membrane interactions of buforin II are related to lipid composition. In this study, we used molecular dynamics (MD) simulations along with lipid vesicle experiments to gain insight into how buforin II interacts differently with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) lipids. Fluorescent spectroscopic measurements agreed with the previous assertion that buforin II does not interact with pure PC vesicles. Nonetheless, the reduced entry of the peptide into anionic PG membranes versus neutral PC membranes during simulations correlates with the experimentally observed reduction in BF2 translocation through pure PG membranes. Simulations showing membrane entry into PC also provide insight into how buforin II may initially penetrate cell membranes. Our MD simulations also allowed us to consider how neutral PE lipids affect the peptide differently than PC. In particular, the peptide had a more helical secondary structure in simulations with PE lipids. A change in structure was also apparent in circular dichroism measurements. PE also reduced membrane entry in simulations, which correlates with decreased translocation in the presence of PE observed in previous studies. Together, these results provide molecular-level insight into how lipid composition can affect buforin II structure and function and will be useful in efforts to design peptides with desired antimicrobial and cell-penetrating properties.     

Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes

The binding of the amphiphilic, positively charged, cyclic beta-sheet antimicrobial decapeptide gramicidin S (GS) to various lipid bilayer model membrane systems was studied by isothermal titration calorimetry. Large unilamellar vesicles composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine or the anionic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, or a binary mixture of the two, with or without cholesterol, were used to mimic the lipid compositions of the outer monolayers of the lipid bilayers of mammalian and bacterial membranes, respectively. Dynamic light scattering results suggest the absence of major alterations in vesicle size or appreciable vesicle fusion upon the binding of GS to the lipid vesicles under our experimental conditions. The binding isotherms can be reasonably well described by a one-site binding model. GS is found to bind with higher affinity to anionic phosphatidylglycerol than to zwitterionic phosphatidylcholine vesicles, indicating that electrostatic interactions in the former system facilitate peptide binding. However, the presence of cholesterol reduced binding only slightly, indicating that the binding of GS is not highly sensitive to the order of the phospholipid bilayer system. Similarly, the measured positive endothermic binding enthalpy (DeltaH) varies only modestly (2.6 to 4.4 kcal/mol), and the negative free energy of binding (DeltaG) also remains relatively constant (-10.9 to -12.1 kcal/mol). The relatively large but invariant positive binding entropy, reflected in relatively large TDeltaS values (13.4 to 16.4 kcal/mol), indicates that GS binding to phospholipid bilayers is primarily entropy driven. Finally, the relative binding affinities of GS for various phospholipid vesicles correlate relatively well with the relative lipid specificity for GS interactions with bacterial and erythrocyte membranes observed in vivo.     

Interdomain and membrane interactions of CTP:phosphocholine cytidylyltransferase revealed via limited proteolysis and mass spectrometry

CTP:phosphocholine cytidylyltransferase (CCT) is a multi-domain enzyme that regulates phosphatidylcholine synthesis. It converts to an active form upon binding cell membranes, and interdomain dissociations have been hypothesized to accompany this process. To identify these interdomain and membrane interactions, the tertiary structures of three forms of CCTalpha were probed by monitoring accessibility to proteases. Time-limited digestion with chymotrypsin or arginine C of soluble CCTalpha (CCT(sol)), phospholipid vesicle-bound CCT (CCT(mem)), and a soluble constitutively active CCT truncated at amino acid 236 generated complex mixtures of peptides that were resolved and identified by gel electrophoresis/immunoblotting and by matrix-assisted laser desorption/ionization-mass spectrometry, with or without coupling to capillary liquid chromatography. Identification of cleavage sites enabled assembly of peptide bond accessibility maps for each CCT form. Our results reveal a approximately 80-residue core within the catalytic domain (domain C) as the most inaccessible region in all three forms and the C-terminal phosphorylation domain as the most accessible. Membrane binding has little effect on the protease accessibility of these domains. To map the protease sites onto the catalytic domain, its three-dimensional structure was modeled from the atomic coordinates of glycerol-phosphate cytidylyltransferase (Protein Data Bank code 1COZ). The protease inaccessibility of most sites in domain C could be explained by burial or location within secondary structural elements. The accessibility of the N-terminal domain (domain N) was enhanced upon membrane binding. Residues Phe(234)-Leu(303) were inaccessible in CCT(mem), suggesting burial in the membrane. Surprisingly, residues Leu(274)-Leu(303) of this domain were also inaccessible in CCT(sol). We propose that this region is buried by interdomain contacts with domain N in CCT(sol). Membrane binding and burial of domain M in the lipid bilayer may disrupt this interaction, leading to increased exposure of sites in domain N.     

Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface.     

Squalamine is not a proton ionophore

Squalamine, an aminosterol antibiotic isolated from the dogfish shark, creates relatively large defects in phospholipid bilayers, allowing the unrestricted translocation of small molecules across these compromised membranes (B.S. Selinsky, Z. Zhou, K.G. Fotjik, S. R. Jones, N.R. Dollahon, A.E. Shinnar, Biochim. Biophys. Acta 1370 (1998) 218-234). However, an aminosterol structurally similar to squalamine was found to act as a proton ionophore in anionic phospholipid vesicles. In contrast with squalamine, gross membrane disruption was not observed with this synthetic analog (G. Deng, T. Dewa, S.L. Regen, J. Am. Chem. Soc. 118 (1996) 8975-8976). In this report, the ionophoric activity of squalamine was tested in anionic and zwitterionic phospholipid vesicles. No ionophoric activity was observed for squalamine in vesicles comprised of phosphatidylglycerol (PG), phosphatidylcholine (PC), or a mixture of the two lipids. Experiments using radiolabeled squalamine indicated that all of the squalamine added to PG vesicles remained with the vesicles, while approximately one-half of the squalamine added to PC vesicles was incorporated. We have synthesized the aminosterol analog of squalamine possessing ionophoric activity, and its ionophoric activity in PG vesicles was confirmed. The synthetic compound possessed no measurable lytic activity when added to preformed phospholipid vesicles. As both compounds possess significant antimicrobial activity, these results suggest that either multiple mechanisms for the antimicrobial activity of aminosterols exist, depending upon the aminosterol structure, or possibly an unrelated common mechanism for antimicrobial activity remains to be discovered.     

Calmodulin binds and stabilizes the regulatory enzyme, CTP: phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCTalpha) is a proteolytically sensitive enzyme essential for production of phosphatidylcholine, the major phospholipid of animal cell membranes. The molecular signals that govern CCTalpha protein stability are unknown. An NH(2)-terminal PEST sequence within CCTalpha did not serve as a degradation signal for the proteinase, calpain. Calmodulin (CaM) stabilized CCTalpha from calpain proteolysis. Adenoviral gene transfer of CaM in cells protected CCTalpha, whereas CaM small interfering RNA accentuated CCTalpha degradation by calpains. CaM bound CCTalpha as revealed by fluorescence resonance energy transfer and two-hybrid analysis. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Gln(243), whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Gln(243) not only resulted in loss of CaM binding but also led to complete calpain resistance in vitro and in vivo. Thus, calpains and CaM both access CCTalpha using a structurally similar molecular signature that profoundly affects CCTalpha levels. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under proinflammatory stress.     

Kinetic study of the aggregation and lipid mixing produced by alpha-sarcin on phosphatidylglycerol and phosphatidylserine vesicles: stopped-flow light scattering and fluorescence energy transfer measurements.

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ammodytoxins, potent presynaptic neurotoxins, are also highly efficient phospholipase A2 enzymes

The enzymatic activity of ammodytoxins (Atxs), secreted phospholipases A(2) (sPLA(2)s) in snake venom, is essential for expression of their presynaptic neurotoxicity, but its exact role in the process is unknown. We have analyzed in detail the enzymatic properties of Atxs, their mutants, and homologues. The apparent rates of phospholipid hydrolysis by the sPLA(2)s tested vary by up to 4 orders of magnitude, and all enzymes display a strong preference for vesicles containing anionic phospholipids, phosphatidylglycerol or phosphatidylserine (PS), over those containing zwitterionic phosphatidylcholine (PC). Nevertheless, Atxs are quite efficient in hydrolyzing pure PC vesicles as well as PC-rich plasma membranes of intact HEK293 cells. The presence of anionic phospholipids in PC vesicles dramatically increases the interfacial binding affinity and catalytic activity of Atxs, but not of their nontoxic homologue ammodytin I(2), that displays unusually low binding affinity and enzymatic activity on PS-containing vesicles and HEK293 plasma membranes. Aromatic and hydrophobic residues on the interfacial binding surface of Atxs are important for productive binding to both zwitterionic and anionic vesicles, while basic and polar residues have a negative impact on binding to zwitterionic vesicles. When tightly bound to the membrane interface, Atxs can reach full enzymatic activity at low micromolar concentrations of Ca(2+). Although Atxs have evolved to function as potent neurotoxins that specifically target presynaptic nerve terminals, they display a high degree of phospholipolytic efficiency on various phospholipid membranes.     

15-deoxy-Delta12,14-prostaglandin J2 impairs phosphatidylcholine synthesis and induces nuclear accumulation of thiol-modified cytidylyltransferase

Synthesis of phosphatidylcholine, the major phospholipid of animal cell membranes, requires the key enzyme cytidylyltransferase (CCTalpha). Cysteine sulfhydryls within CCTalpha are needed for full catalytic activity. Here we show that prostaglandin 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) inactivates CCTalpha by inducing generation of reactive oxidant species and the appearance of a cross-linked CCTalpha dimer in cells. N-Acetyl-l-cysteine reduced oxidative stress, prevented CCTalpha cross-linking, and restored CCT function in 15d-PGJ(2)-treated cells. 15d-PGJ(2) modified critical cysteine residues within CCTalpha as determined by mutagenesis studies and by incorporation of biotin-15d-PGJ(2) into CCTalpha. These effects of 15d-PGJ(2) were associated with CCTalpha accumulation within the nucleus. The data indicate that bioactive prostanoids significantly impair membrane phospholipid production by promoting cysteine cross-bridging within CCTalpha.     

Effects of proteins on phospholipid vesicle aggregation and lipid vesicle-monolayer interactions

The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca²⁺-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca²⁺-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca²⁺-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes.     

Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.     

Increased rates of lipid exchange between Mycoplasma capricolum membranes and vesicles in relation to the propensity of forming nonbilayer lipid structures

We have studied the effects of modification of the endogenous phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) content of the plasma membrane of Mycoplasma capricolum on the kinetics of spontaneous [14C]cholesterol and 14C-labeled phospholipid exchange between M. capricolum membranes and lipid vesicles. The PG/DPG molar ratio of M. capricolum membranes changed when cells were grown in media supplemented with 0.5 mM CaCl2 and/or egg phosphatidylcholine (PC) (10-20 micrograms/ml), increasing from 3.9 to 6.3 on supplementation with Ca2+; this ratio decreased to 1.1 in media supplemented with PC and to 1.8 in media containing both PC and Ca2+. The ratio of palmitate to oleate in both PG and DPG decreased when cells were grown with PC or with PC and Ca2+. Bilayer disruptions were seen in freeze-fracture electron micrographs of trypsin-treated M. capricolum membranes from cells grown with both Ca2+ and PC, and numerous lipidic particles and other bilayer disruptions were observed in trypsin-treated M. capricolum membranes and their lipid extracts. The rates of spontaneous exchange of 14C-labeled cholesterol and PC from membranes isolated from cells grown with PC and Ca2+ to acceptor lipid vesicles were exchanged by approximately 30%, and the rate of the rapidly exchangeable cholesterol pool in intact cells was enhanced by 64%. The enhancements in cholesterol and PC exchange rates are considered to result from structural defects expected in the M. capricolum membranes obtained from cells grown with Ca2+ supplementation. Our findings parallel previous examples of functional modifications of membranes induced by bilayer instability arising from a pretransitional state leading to the onset of a nonlamellar phase.     

Reversible Liposome Association Induced by LAH4: A Peptide with Potent Antimicrobial and Nucleic Acid Transfection Activities

We report on the reversible association of anionic liposomes induced by an antimicrobial peptide (LAH4). The process has been characterized for mixed membranes of POPC and POPS at molar ratios of 1:1, 3:1, and 9:1. Although the vesicles remain in suspension in the presence of excess amounts of peptide, the addition of more lipids results in surface charge neutralization, aggregation of the liposomes, and formation of micrometer-sized structures that coexist in equilibrium with vesicles in suspension. At low ratios of anionic lipids, vesicle aggregation is a reversible process, and vesicle disassembly is observed upon inversion of the surface charge by further supplementation with anionic vesicles. In contrast, a different process, membrane fusion, occurs in the presence of high phosphatidylserine concentrations. Upon binding to membranes containing low POPS concentrations, the peptide adopts an in-plane α-helical structure, a secondary structure that is conserved during vesicle association and dissociation. Our finding that peptides are essential for vesicle aggregation contributes to a better understanding of the activity of antimicrobial peptides, and suggests an additional layer of complexity in membrane-protein lipid interactions.