Translocation of protegrin I through phospholipid membranes: role of peptide folding

The protegrin PG-1, belonging to the family of β-stranded antimicrobial peptides, exerts its activity by forming pores in the target biological membranes. Linear analogues derived from PG-1 do not form pores in the phospholipid membranes and have been used successfully to deliver therapeutic compounds into eucaryotic cells. In this paper, the translocation of PG-1 and of a linear analogue through artificial phospholipid membranes was investigated, leading to a possible mechanism for the activity of these peptidic vectors. We report here that [12W]PG-1, a fluorescent analogue of PG-1, is able to translocate through lipid bilayers and we demonstrate that this property depends on its secondary structure. Our results agree with the recent mechanism proposed for the translocation and permeabilisation activities of several helical and β-stranded peptides. In addition, our data corroborate recent work suggesting that certain protegrin-derived vectors enter into endothelial cells by adsorptive-mediated endocytosis.     

Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.     

Structures of β-hairpin antimicrobial protegrin peptides in lipopolysaccharide membranes: mechanism of gram selectivity obtained from solid-state nuclear magnetic resonance

The structural basis for the gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy. The hexa-arginine PG-1 exhibits potent activities against both gram-positive and gram-negative bacteria, while a mutant of PG-1 with only three cationic residues maintains gram-positive activity but is 30-fold less active against gram-negative bacteria. We determined the topological structure and lipid interactions of these two peptides in a lipopolysaccharide (LPS)-rich membrane that mimics the outer membrane of gram-negative bacteria and in the POPE/POPG membrane, which mimics the membrane of gram-positive bacteria. (31)P NMR line shapes indicate that both peptides cause less orientational disorder in the LPS-rich membrane than in the POPE/POPG membrane. (13)C chemical shifts and (13)C-(1)H dipolar couplings show that both peptides maintain their β-hairpin conformation in these membranes and are largely immobilized, but the mutant exhibits noticeable intermediate-time scale motion in the LPS membrane at physiological temperature, suggesting shallow insertion. Indeed, (1)H spin diffusion from lipid chains to the peptides shows that PG-1 fully inserts into the LPS-rich membrane whereas the mutant does not. The (13)C-(31)P distances between the most hydrophobically embedded Arg of PG-1 and the lipid (31)P are significantly longer in the LPS membrane than in the POPE/POPG membrane, indicating that PG-1 does not cause toroidal pore defects in the LPS membrane, in contrast to its behavior in the POPE/POPG membrane. Taken together, these data indicate that PG-1 causes transmembrane pores of the barrel-stave type in the LPS membrane, thus allowing further translocation of the peptide into the inner membrane of gram-negative bacteria to kill the cells. In comparison, the less cationic mutant cannot fully cross the LPS membrane because of weaker electrostatic attractions, thus causing weaker antimicrobial activities. Therefore, strong electrostatic attraction between the peptide and the membrane surface, ensured by having a sufficient number of Arg residues, is essential for potent antimicrobial activities against gram-negative bacteria. The data provide a rational basis for controlling gram selectivity of AMPs by adjusting the charge densities.     

Hexadecylphosphocholine inhibits translocation of CTP:choline-phosphate cytidylyltransferase in Madin-Darby canine kidney cells.

The mechanism of the inhibition of phosphatidylcholine biosynthesis by the phospholipid analogue, hexadecylphosphocholine, was investigated in Madin-Darby canine kidney cells. In the presence of 50 mumol/liter hexadecylphosphocholine, there was a translocation of CTP:choline-phosphate cytidylyltransferase (EC 22.7.7.15) activity from the membranes to the cytosol of the cells. Since we recently demonstrated that hexadecylphosphocholine also inhibits protein kinase C in vitro, [methyl-3H]choline labeling experiments were repeated with phorbol ester-desensitized cells. In these cells the same inhibitory effect of hexadecylphosphocholine was measured. As a consequence of inhibition, the [methyl-3H]choline incorporation into the phosphocholine pool was increased time-dependently. In addition, there was no evidence for a difference between the choline uptake of control and hexadecylphosphocholine-treated cells. Likewise, the amount of diacylglycerol, a known activator of the translocation process, was not reduced. Finally, we showed that the inhibitory effect of hexadecylphosphocholine on CTP:choline-phosphate cytidylyltransferase translocation cannot be explained by the detergent properties of this phospholipid analogue. Therefore, we suggest a direct inhibitory effect of hexadecylphosphocholine on the translocation of CTP:choline-phosphate cytidylyltransferase.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a beta-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.     

Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among β-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a β-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the β-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.     

Translocation of the pAntp peptide and its amphipathic analogue AP-2AL

The pAntp peptide, corresponding to the third helix of the homeodomain of the Antennapedia protein, enters by a receptor-independent process into eukaryotic cells. The interaction between the pAntp peptide and the phospholipid matrix of the plasma membrane seems to be the first step involved in the translocation mechanism. However, the mechanism by which the peptide translocates through the cell membrane is still not well established. We have investigated the translocation ability of pAntp through a protein-free phospholipid membrane in comparison with a more amphipathic analogue. We show by fluorescence spectroscopy, circular dichroism, NMR spectroscopy, and molecular modeling that pAntp is not sufficiently helically amphipathic to cross a phospholipid membrane of a model system. Due to its primary sequence related to its DNA binding ability in the Antennapedia homeodomain-DNA complex, the pAntp peptide does not belong to the amphipathic alpha-helical peptide family whose members are able to translocate by pore formation.     

Lipid domain separation,bilayer thickening and pearling induced by the cell penetrating peptide penetratin

Protein membrane transduction domains are able to translocate through cell membranes. This capacity resulted in new concepts on cell communication and in the design of vectors for internalization of active molecules into cells. Penetratin crosses the plasma membrane by a receptor and metabolic energy-independent mechanism which is at present unknown. A better knowledge of its interaction with phospholipids will help to understand the molecular mechanisms of cell penetration. Here, we investigated the role of lipid composition on penetratin induced membrane perturbations by X-ray diffraction, microscopy and ³¹P-NMR. Penetratin showed the ability to induce phospholipid domain separation, membrane bilayer thickening, formation of vesicles, membrane undulations and tubular pearling. These data demonstrate its capacity to increase membrane curvature and suggest that dynamic phospholipid–penetratin complexes can be organized in different structural arrangements. These properties and their implications in peptide membrane translocation capacity are discussed.     

Headgroup structure and fatty acid chain length of the acidic phospholipids modulate the interaction of membrane mimetic vesicles with the antimicrobial peptide protegrin-1.

The interaction of protegrin-1 (PG-1), a small beta-sheet antimicrobial peptide with acidic phospholipid model membranes was investigated by differential scanning calorimetry. We found that PG-1 can distinguish between liposomes of the anionic phospholipids DPPG, DPPS and DPPA, eventhough the headgroups of these phospholipids all have the same net charge and they carry the same hydrocarbon chains. Specifically, PG-1 had only a minor effect on the thermotropic phase behavior of DPPA liposomes, while it interacted preferentially with the fluid phase of DPPS. Furthermore, PG-1 could induce a phase separation in DPPG liposomes resulting in the formation of peptide-rich domains even at low concentrations of the peptide. However, this peptide-rich domain was not evident when the fatty acyl chains were longer or shorter by two carbon atoms. In addition, PG-1 can also form peptide-rich domains in DPPS vesicles but only at high concentrations of the peptide. These results suggest that in addition to an overall negative charge, the structural features of the phospholipid headgroups, lipid packing and thus membrane fluidity will influence the interaction with PG-1, thereby modulating its biological activity.     

Membrane interactions of antimicrobial peptides from Australian tree frogs

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic alpha-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.     

Mechanism of structural transformations induced by antimicrobial peptides in lipid membranes

It has long been suggested that pore formation is responsible for the increase in membrane permeability by antimicrobial peptides (AMPs). To better understand the mechanism of AMP activity, the disruption of model membrane by protegrin-1 (PG-1), a cationic antimicrobial peptide, was studied using atomic force microscopy. We present here the direct visualization of the full range of structural transformations in supported lipid bilayer patches induced by PG-1 on zwitterionic 1,2-dimyristoyl-snglycero-phospho-choline (DMPC) membranes. When PG-1 is added to DMPC, the peptide first induces edge instability at low concentrations, then pore-like surface defects at intermediate concentrations, and finally wormlike structures with a specific length scale at high concentrations. The formation of these structures can be understood using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent. Atomistic molecular dynamics simulations on lipid bilayer ribbons with PG-1 molecules placed at the edge or interior positions are carried out to calculate the effect of PG-1 in reducing line tension. Further investigation of the placement of PG-1 and its association with defects in the bilayer is carried out using unbiased assembly of a PG-1 containing bilayer from a random mixture of PG-1, DMPC, and water. A generalized model of AMP induced structural transformations is also presented in this work. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Membrane interactions of antimicrobial peptides from Australian tree frogs

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic α-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.     

Polar angle as a determinant of amphipathic alpha-helix-lipid interactions: a model peptide study

Various physicochemical properties play important roles in the membrane activities of amphipathic antimicrobial peptides. To examine the effects of the polar angle, two model peptides, thetap100 and thetap180, with polar angles of 100 degrees and 180 degrees, respectively, were designed, and their interactions with membranes were investigated in detail. These peptides have almost identical physicochemical properties except for polar angle. Like naturally occurring peptides, these peptides selectively bind to acidic membranes, assuming amphipathic alpha-helices, and formed peptide-lipid supramolecular complex pores accompanied by lipid flip-flop and peptide translocation. Despite its somewhat lower membrane affinity, thetap100 exhibited higher membrane permeabilization activity, a greater flip-flop rate, as well as more antimicrobial activity due to a higher pore formation rate compared with thetap180. Consistent with these results, the peptide translocation rate of thetap100 was higher. Furthermore, the number of peptides constituting thetap100 pores was less than that of thetap180, and thetap100 pores involved more lipid molecules, as reflected by its cation selectivity. The polar angle was found to be an important parameter determining peptide-lipid interactions.     

Beyond electrostatics: Antimicrobial peptide selectivity and the influence of cholesterol-mediated fluidity and lipid chain length on protegrin-1 activity

Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1’s incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents.     

Membrane-disruptive abilities of β-hairpin antimicrobial peptides correlate with conformation and activity: A P and H NMR study

The membrane interaction and solution conformation of two mutants of the β-hairpin antimicrobial peptide, protegrin-1 (PG-1), are investigated to understand the structural determinants of antimicrobial potency. One mutant, [A_6,8,13,15] PG-1, does not have the two disulfide bonds in wild-type PG-1, while the other, [Δ_4,18 G₁₀] PG-1, has only half the number of cationic residues. ³¹P solid-state NMR lineshapes of uniaxially aligned membranes indicate that the membrane disorder induced by the three peptides decreases in the order of PG-1>[Δ_4,18 G₁₀] PG-1?[A_6,8,13,15] PG-1. Solution NMR studies of the two mutant peptides indicate that [Δ_4,18 G₁₀] PG-1 preserves the β-hairpin fold of the wild-type peptide while [A_6,8,13,15] PG-1 adopts a random coil conformation. These NMR results correlate well with the known activities of these peptides. Thus, for this class of peptides, the presence of a β-hairpin fold is more essential than the number of cationic charges for antimicrobial activity. This study indicates that ³¹P NMR lineshapes of uniaxially aligned membranes are well correlated with antimicrobial activity, and can be used as a diagnostic tool to understand the peptide-lipid interactions of these antimicrobial peptides.     

The effect of cyclization of magainin 2 and melittin analogues on structure, function, and model membrane interactions: implication to their mode of action

The amphipathic alpha-helical structure is a common motif found in membrane binding polypeptides including cell lytic peptides, antimicrobial peptides, hormones, and signal sequences. Numerous studies have been undertaken to understand the driving forces for partitioning of amphipathic alpha-helical peptides into membranes, many of them based on the antimicrobial peptide magainin 2 and the non-cell-selective cytolytic peptide melittin, as paradigms. These studies emphasized the role of linearity in their mode of action. Here we synthesized and compared the structure, biological function, and interaction with model membranes of linear and cyclic analogues of these peptides. Cyclization altered the binding of melittin and magainin analogues to phospholipid membranes. However, at similar bound peptide:lipid molar ratios, both linear and cyclic analogues preserved their high potency to permeate membranes. Furthermore, the cyclic analogues preserved approximately 75% of the helical structure of the linear peptides when bound to membranes. Biological activity studies revealed that the cyclic melittin analogue had increased antibacterial activity but decreased hemolytic activity, whereas the cyclic magainin 2 analogue had a marked decrease in both antibacterial and hemolytic activities. The results indicate that the linearity of the peptides is not essential for the disruption of the target phospholipid membrane, but rather provides the means to reach it. In addition, interfering with the coil-helix transition by cyclization, while maintaining the same sequence of hydrophobic and positively charged amino acids, allows a separated evaluation of the hydrophobic and electrostatic contributions to binding of peptides to membranes.     

Insights into buforin II membrane translocation from molecular dynamics simulations

Buforin II is a histone-derived antimicrobial peptide that readily translocates across lipid membranes without causing significant membrane permeabilization. Previous studies showed that mutating the sole proline of buforin II dramatically decreases its translocation. As well, researchers have proposed that the peptide crosses membranes in a cooperative manner by forming transient toroidal pores. This paper reports molecular dynamics simulations designed to investigate the structure of buforin II upon membrane entry and evaluate whether the peptide is able to form toroidal pore structures. These simulations showed a relationship between protein–lipid interactions and increased structural deformations of the buforin N-terminal region promoted by proline. Moreover, simulations with multiple peptides show how buforin II can embed deeply into membranes and potentially form toroidal pores. Together, these simulations provide structural insight into the translocation process for buforin II in addition to providing more general insight into the role proline can play in antimicrobial peptides.     

Parallel and antiparallel dimers of magainin 2: their interaction with phospholipid membrane and antibacterial activity.

Magainin 2 (M2) forms pores by associating with several other M2 molecules in lipid membranes and shows antibacterial activity. To examine the effect of M2 dimerization on biological activity and membrane interaction, parallel and antiparallel M2 dimers were prepared from two monomeric precursors. Antibacterial and haemolytic activities were enhanced by dimerization. CD measurements showed that both dimers and monomers have an alpha-helical structure in the presence of lipid vesicles. Tryptophan fluorescence shift and KI quenching studies showed that all the peptides were more deeply embedded in acidic liposomes than in neutral liposomes. Experiments on dye-leakage activity and membrane translocation of peptides suggest that dimers and monomers form pores through lipid membranes, although the pore formation may be accompanied by membrane disturbance. Although dimerization of M2 increased the interaction activity with lipid membranes, no appreciable difference between the activities of parallel and antiparallel M2 dimers was observed.     

Secondary structure of cell-penetrating peptides controls membrane interaction and insertion

The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: Effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a β-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.