The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and 31P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the (31)P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. (2)H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. (31)P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, (31)P and (2)H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the ³¹P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. ²H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. ³¹P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, ³¹P and ²H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

A 2H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

A H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

Interaction of phosphatidylserine synthase from E. coli with lipid bilayers: coupled plasmon-waveguide resonance spectroscopy studies

The interaction of phosphatidylserine (PS) synthase from Escherichia coli with lipid membranes was studied with a recently developed variant of the surface plasmon resonance technique, referred to as coupled plasmon-waveguide resonance spectroscopy. The features of the new technique are increased sensitivity and spectral resolution, and a unique ability to directly measure the structural anisotropy of lipid and proteolipid films. Solid-supported lipid bilayers with the following compositions were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (80:20, mol/mol); POPC-POPA (60:40, mol/mol); and POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (75:25, mol/mol). Addition of either POPA or POPG to a POPC bilayer causes a considerable increase of both the bilayer thickness and its optical anisotropy. PS synthase exhibits a biphasic interaction with the bilayers. The first phase, occurring at low protein concentrations, involves both electrostatic and hydrophobic interactions, although it is dominated by the latter, and the enzyme causes a local decrease of the ordering of the lipid molecules. The second phase, occurring at high protein concentrations, is predominantly controlled by electrostatic interactions, and results in a cooperative binding of the enzyme to the membrane surface. Addition of the anionic lipids to a POPC bilayer causes a 5- to 15-fold decrease in the protein concentration at which the first binding phase occurs. The results reported herein lend experimental support to a previously suggested mechanism for the regulation of the polar head group composition in E. coli membranes.     

Insertion of Dengue E into lipid bilayers studied by neutron reflectivity and molecular dynamics simulations

The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and target membranes, which is essential for infectivity. Insertion of E into the target membrane serves to anchor E and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, orientation of the trimer with respect to the membrane normal, and the interactions that form between trimer and membrane. In the present work, we resolved the depth of insertion, the tilt angle, and the fundamental interactions for the soluble portion of Dengue E trimers (sE) associated with planar lipid bilayer membranes of various combinations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and cholesterol (CHOL) by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The results show that the tip of E containing the fusion loop (FL) is located at the interface of the headgroups and acyl chains of the outer leaflet of the lipid bilayers, in good agreement with prior predictions. The results also indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or CHOL. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines and arginines located on the sides of the trimer close to the tip (K246, K247, and R73) and nearby lipid headgroups. These hydrogen bonds provide a major contribution to the membrane anchoring and may help to destabilize the target membrane.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Structure and dynamics of the two amphipathic arginine-rich peptides RW9 and RL9 in a lipid environment investigated by solid-state NMR and MD simulations

Cell penetrating peptides (CPPs) are able to cross membranes without using receptors but only little information about the underlying mechanism is available. In this work, we investigate the interaction of the two arginine-rich CPPs RW9 and RL9 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), and POPC/POPG membranes with varying POPG content using isothermal titration calorimetry (ITC), solid-state nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics (MD) simulations. Both peptides were derived from the known CPP penetratin and it was shown previously that RW9 is able to penetrate membranes better than RL9. Overall, the results show that both RW9 and RL9 have a relatively small influence on the membrane. They increase the order of the lipids in the headgroup region and reduce order in the acyl chains indicating that they are located in the lipid/water interface. In addition, the flexibility of the membrane is slightly increased by both peptides but RW9 has a larger influence than RL9. The differences observed in the influences on POPC and POPG as well as MD simulations on the mixed POPC/POPG bilayers of 850 ns length each show that both peptides preferentially associate with and enrich the charged PG lipids almost 2fold in an area of 12 Å around the peptides. As expected, we could not observe any membrane crossing on the simulation time scale of 850 ns but observed that some peptides flipped their orientation during binding to the membrane. Interestingly, all observed flips coincided with structural changes in the peptides indicating that structural changes or flexibility might play a role during the binding of arginine-rich CPPs to membranes.     

Acyl chain dynamics of phosphatidylethanolamines containing oleic acid and dihydrosterculic acid: 2H NMR relaxation studies

The dynamical behavior of the acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphoethanolamine has been investigated by using 2H T1 and T2 relaxation times. Lipids were labeled at the 5-,9-,10-, and 16-positions of the sn-2 acyl chain. The profile of deuterium spin-lattice relaxation rate (T1(-1) vs. chain position is characterized in all systems by a marked discontinuity at the positions of the carbon-carbon double bond and the cyclopropane ring; the deuterons at these positions have relaxation rates which are greater than at any other labeled position of the sn-2 chain. For both types of sn-2 acyl chain, assuming a single-exponential correlation time and that the motion is within the rapid regime, the phosphatidylcholine lipid systems are less mobile than their phosphatidylethanolamine analogues. Systems containing an oleoyl chain are more dynamic than their analogues containing a dihydrosterculoyl chain. The rates of motion of the sn-2 acyl chains of phosphatidylethanolamine in a bilayer structure are slower than those of the lipid in an inverted hexagonal structure. In the hexagonal phase, the motional rates of a dihydrosterculoyl chain are slower than those of the corresponding positions of an oleoyl chain.     

A coarse-grained approach to studying the interactions of the antimicrobial peptides aurein 1.2 and maculatin 1.1 with POPG/POPE lipid mixtures

In the present work we investigated the differential interactions of the antimicrobial peptides (AMPs) aurein 1.2 and maculatin 1.1 with a bilayer composed of a mixture of the lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). We carried out molecular dynamics (MD) simulations using a coarse-grained approach within the MARTINI force field. The POPE/POPG mixture was used as a simple model of a bacterial (prokaryotic cell) membrane. The results were compared with our previous findings for structures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a representative lipid of mammalian cells. We started the simulations of the peptide–lipid system from two different initial conditions: peptides in water and peptides inside the hydrophobic core of the membrane, employing a pre-assembled lipid bilayer in both cases. Our results show similarities and differences regarding the molecular behavior of the peptides in POPE/POPG in comparison to their behavior in a POPC membrane. For instance, aurein 1.2 molecules can adopt similar pore-like structures on both POPG/POPE and POPC membranes, but the peptides are found deeper in the hydrophobic core in the former. Maculatin 1.1 molecules, in turn, achieve very similar structures in both kinds of bilayers: they have a strong tendency to form clusters and induce curvature. Therefore, the results of this study provide insight into the mechanisms of action of these two peptides in membrane leakage, which allows organisms to protect themselves against potentially harmful bacteria.

Open image in new window

Graphical Abstract Aurein pore structure (green) in a lipid bilayer composed by POPE (blue) and POPG (red) mixture. It is possible to see water beads (light blue) inside the pore.

     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Comparative study of stability and transport of molecules through cyclic peptide nanotube and aquaporin: a molecular dynamics simulation approach

Abstract

The structural stability and transport properties of the cyclic peptide nanotube (CPN) 8?×?[Cys–Gly–Met–Gly]₂ in different phospholipid bilayers such as POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) and POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine) with water have been investigated using molecular dynamics (MD) simulation. The hydrogen bonds and non-bonded interaction energies were calculated to study the stability in different bilayers. One µs MD simulation in POPA lipid membrane reveals the stability of the cyclic peptide nanotube, and the simulations at various temperatures manifest the higher stability of 8?×?[Cys–Gly–Met–Gly]₂. We demonstrated that the presence of sulphur-containing amino acids in CPN enhances the stability through disulphide bonds between the adjacent rings. Further, the water permeation coefficient of the CPN is calculated and compared with human aquaporin-2 (AQP2) channel protein. It is found that the coefficients are highly comparable to the AQP2 channel though the mechanism of water transport is not similar to AQP 2; the flow of water in the CPN is taking place as a two-line 1–2–1–2 file fashion. In addition to that, the transport behavior of Na⁺ and K⁺ ions, single water molecule, urea and anti-cancer drug fluorouracil were investigated using pulling simulation and potential of mean force calculation. The above transport behavior shows that Na⁺ is trapped in CPN for a longer time than other molecules. Also, the interactions of the ions and molecules in Cα and mid-Cα plane were studied to understand the transport behavior of the CPN. Abbreviations AQP2 Aquaporin-2

CPN Cyclic peptide nanotube

MD Molecular dynamics

POPA 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid

POPE 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

POPG 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol

POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine

Communicated by Ramaswamy H. Sarma     

Phosphatidylethanolamine-phosphatidylglycerol bilayer as a model of the inner bacterial membrane

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the main lipid components of the inner bacterial membrane. A computer model for such a membrane was built of palmitoyloleoyl PE (POPE) and palmitoyloleoyl PG (POPG) in the proportion 3:1, and sodium ions (Na+) to neutralize the net negative charge on each POPG (POPE-POPG bilayer). The bilayer was simulated for 25 ns. A final 10-ns trajectory fragment was used for analyses. In the bilayer interfacial region, POPEs and POPGs interact readily with one another via intermolecular hydrogen (H) bonds and water bridges. POPE is the main H-bond donor in either PEPE or PEPG H-bonds; PGPG H-bonds are rarely formed. Almost all POPEs are H-bonded and/or water bridged to either POPE or POPG but PE-PG links are favored. In effect, the atom packing in the near-the-interface regions of the bilayer core is tight. Na+ does not bind readily to lipids, and interlipid links via Na+ are not numerous. Although POPG and POPE comprise one bilayer, their bilayer properties differ. The average surface area per POPG is larger and the average vertical location of the POPG phosphate group is lower than those of POPE. Also, the alkyl chains of POPG are more ordered and less densely packed than the POPE chains. The main conclusion of this study is that in the PE-PG bilayer PE interacts more strongly with PG than with PE. This is a likely molecular-level event behind a regulating mechanism developed by the bacteria to control its membrane permeability and stability consisting in changes of the relative PG/PE concentration in the membrane.     

Algorithm to catalogue topologies of dynamic lipid hydrogen-bond networks

Lipid membrane interfaces host reactions essential for the functioning of cells. The hydrogen-bonding environment at the membrane interface is particularly important for binding of proteins, drug molecules, and ions. We present here the implementation and applications of a depth-first search algorithm that analyzes dynamic lipid interaction networks. Lipid hydrogen-bond networks sampled transiently during simulations of lipid bilayers are clustered according to main types of topologies that characterize three-dimensional arrangements of lipids connected to each other via short water bridges. We characterize the dynamics of hydrogen-bonded lipid clusters in simulations of model POPE and POPE:POPG membranes that are often used for bacterial membrane proteins, in a model of the Escherichia coli membrane with six different lipid types, and in POPS membranes. We find that all lipids sample dynamic hydrogen-bonded networks with linear, star, or circular arrangements of the lipid headgroups, and larger networks with combinations of these three types of topologies. Overall, linear lipid-water bridges tend to be short. Water-mediated lipid clusters in all membranes with PE lipids tend to be somewhat small, with about four lipids in all membranes studied here. POPS membranes allow circular arrangements of three POPS lipids to be sampled frequently, and complex arrangements of linear, star, and circular paths may also be sampled. These findings suggest a molecular picture of the membrane interface whereby lipid molecules transiently connect in clusters with somewhat small spatial extension.     

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 degrees C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 degrees C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.     

Construction and optimisation of a computer model for a bacterial membrane

The main steps in the construction of a computer model for a bacterial membrane are described. The membrane has been built of 72 lipid molecules, 54 of which being 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine (POPE) and 18--1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidyl-rac-glycerol (POPG) molecules (thus in the proportion of 3:1). The membrane was hydrated with 1955 water molecules (approximately 27 water molecules per lipid). To neutralise the electronic charge (-e) on each POPG molecule, 18 sodium ions (Na+) were added to the membrane close to the POPG phosphate groups. The atomic charges on the POPE and POPG headgroups were obtained from ab initio quantum mechanical restrained electrostatic potential fitting (RESP) (Bayly et al., 1993, J. Phys. Chem. 97, 10269) using the GAMESS program at the 6-31G* level (Schmidt et al., 1993, J. Comput. Chem. 14, 1347). The model constructed in this way provided an initial structure for subsequent molecular dynamics simulation studies intended to elucidate the atomic level interactions responsible for the structure and dynamics of the bacterial membrane.     

Effects of replacement of a double bond by a cyclopropane ring in phosphatidylethanolamines: a 2H NMR study of phase transitions and molecular organization

The thermotropic behavior and molecular properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphoethanolamine (PDSPE) have been investigated by 2H NMR spectroscopy using samples selectively labeled at the 5'-, 9'-, 10'-, and 16'-positions of the sn-2 chains. Comparison with the corresponding phosphocholine analogues (POPC and PDSPC), obtained as intermediate synthetic products, was used to monitor the role of the polar head group. Replacement of the choline moiety by ethanolamine increased the gel to liquid-crystal transition temperature by 10-32 degrees C and led to a significantly higher ordering of the fatty acyl chains in the liquid-crystalline bilayer state. The lateral compression effect, due to the smaller area per polar head group in PE, results in a bilayer to hexagonal phase transition at elevated temperatures. The effects on both PC and PE due to replacement of the olefinic group by a cyclopropane unit are similar. A decrease in the temperature of the gel to liquid-crystal phase transition, Tc, is observed upon introduction of a cyclopropane ring; it goes from 26 degrees C in POPE to approximately 10 degrees C in PDSPE. In addition, a very significant broadening of the transition profile is observed. These observations are consistent with the poor packing ability of mixed saturated and cyclopropane-containing chains due to the bulky substituent effect. The temperature of the bilayer-hexagonal phase transition of PE samples was decreased by 15-20 degrees C on replacement of oleoyl chains by dihydrosterculoyl chains at the sn-2 position.(ABSTRACT TRUNCATED AT 250 WORDS)