How Membrane-Active Peptides Get into Lipid Membranes

Biography ... Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer ..... Tóth , Györgyi Váradi , ...
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How Membrane-Active Peptides Get into Lipid Membranes Marc-Antoine Sani and Frances Separovic* School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, Victoria 3010, Australia CONSPECTUS: The structure−function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular mechanism by which these membrane-active peptides lyse membranes. The last class of membrane-active peptides discussed are the CPPs, which translocate across the lipid bilayer without inducing severe disruption and have potential as drug vehicles. CPPs are typically highly charged and can show antimicrobial activity by targeting an intracellular target rather than via a direct membrane lytic mechanism. A critical aspect in the structure−function relationship of membrane-active peptides is their specific activity relative to the lipid membrane composition of the cell target. Cell membranes have a wide diversity of lipids, and those of eukaryotic and prokaryotic species differ greatly in composition and structure. The activity of AMPs from Australian tree frogs, toxins, and CPPs has been investigated within various lipid systems to assess whether a relationship between peptide and membrane composition could be identified. NMR spectroscopy techniques are being used to gain atomistic details of how these membrane-active peptides interact with model membranes and cells, and in particular, competitive assays demonstrate the difference between affinity and activity for a specific lipid environment. Overall, the interactions between these relatively small sized peptides and various lipid bilayers give insight into how these peptides function at the membrane interface.

1. INTRODUCTION Peptides have a diverse range of cellular functions, participate in signaling pathways, display membrane activity as drug carriers or lytic agents, and form complex molecular assemblies with the lipid membrane. In particular, antimicrobial and toxin peptides actively target the lipid membranes of cells to insert and ultimately disrupt their integrity.1−4 The molecular mechanisms are complex and strongly dependent on the lipid composition of the membrane and on the metabolic activity of the cellular target. For instance, antimicrobial peptides (AMPs) are primarily lytic, but recent findings show that they can also act as signaling molecules at sublethal concentrations.5 Figure 1 illustrates a generic peptide assumed to adopt a helical structure when bound to a lipid membrane, and several mechanisms of action are sketched. The diverse function and utility of © XXXX American Chemical Society

membrane-active peptides demonstrate possibilities as a pipeline for drug discovery and drug delivery, which have led to efforts to understand their activity and, in particular, their interactions with cell membranes. These peptides deliver their action within the lipid membrane environment, which also is under continual investigation as new classes of lipids and their functions are revealed; for instance, amino-acylated lipids are emerging as a factor of resistance for Staphylococcus aureus against cationic molecules.6 The composition of cell membranes is highly diverse, which allows design of peptides for optimum cellular activity in a specific lipid environment or targeting of particular organisms. In cases of severe environReceived: February 10, 2016

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DOI: 10.1021/acs.accounts.6b00074 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 1. AMPs are typically unstructured in aqueous solution. In the presence of a lipid membrane, the peptides may assume an α-helical structure and disrupt the bilayer structure. Several modes of action have been proposed in model membranes: the barrel-stave pore, toroidal pore, or carpet mechanism, as well as a number of less disruptive mechanisms. The mechanism is dependent on the peptide and the lipid membrane composition, and the mechanisms of membrane bilayer perturbation may change depending on peptide concentration, temperature, and pH.

Table 1. Structure and Activitya of AMPs and Toxins peptideb

aa

net charge (pH 7)

hydrophobicityc (%)

HC50 (μM)

MIC (μM) E. coli

MIC (μM) S. aureus

cupiennin 1a melittin caerin 1.1 maculatin 1.1 citropin 1.1 aurein 1.2 UyCT5

35 26 25 21 16 13 13

+8 +5 +1 +1 +2 +1 +2

51 54 68 64 56 54 62

24 1 >39 30 >62 >68 14

0.5 2 >39 64 40 >68 32

0.5 0.5 1 4 5 34 2

a See text for references. bPeptide sequences: cupiennin 1a, GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2; melittin, GIGAVLKVLTTGLPALISWIKRKRQQ; caerin 1.1, GLLSVLGSVAKHVLPHVVPVIAEHL-NH2; maculatin 1.1, GLFGVLAKVAAHVVPAIAEHF-NH2; citropin 1.1, GLFDVIKKVASVIGGL-NH2; aurein 1.2, GLFDIIKKIAESF-NH2; UyCT5, IWSAIWSGIKGLL-NH2. cBased on the Hopp−Woods scale.19

identify the structure−function relationship that regulates their membrane activity. These AMPs have similar structural and chemical properties but their antimicrobial activity is dependent on the bacterial species. The interactions behind the specific antimicrobial activity have been investigated using negatively charged vesicles that mimic the membranes of Gram-positive or Gram-negative bacteria. Neutral or zwitterionic vesicles that mimic eukaryotic membranes were also used to assess the mechanisms behind hemolytic activity. The secondary structure and leakage activity of the AMPs and the lipid bilayer response upon peptide binding are reviewed. The molecular mechanisms are compared with those of cytotoxic peptides from bee and

mental change, organisms may even respond by actively changing their membrane lipid composition.7 For instance, in order to maintain the appropriate membrane fluidity for efficient cellular processes, bacteria are able to produce a higher proportion of lipids with unsaturated acyl chains at lower temperatures while saturated chains are the dominant species at higher temperatures. Therefore, understanding the lipid membrane environment as well as the peptide structure is important in the quest to decipher the mode of action of these membrane-active peptides. Four AMPs found on the skin of Australian tree frogs have been studied within various lipid environments in order to B

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Figure 2. NMR solution structures of Australian tree frog AMPs9−12 represented as a Coloumbic surface using the Chimera graphic software. The color code indicates positive (blue set as −5 kcal/mol), neutral (white), and negative (red set as 5 kcal/mol) electrostatic potentials.

comparison of hydrophobicity and membrane lytic activity. Caerin with 68% hydrophobic residues is weakly hemolytic (>38 μM) and melittin, with a similar number of amino acids but lower hydrophobicity of 54%, is highly hemolytic (1 μM).17 Maculatin is four amino acids shorter with 64% hydrophobic residues, citropin is 9 amino acids shorter with 54% hydrophobic residues, and cupiennin at 10 amino acid longer has the lowest hydrophobic content (51%) and is also weakly hemolytic (Table 1).17,18 Since red blood cell membranes are mostly neutral, highly charged peptides are not anticipated to have a severe hemolytic activity. However, melittin is highly cationic (+5) but also highly hemolytic, while the weakly hemolytic caerin and cupiennin are either weakly charged (+1) or highly charged (+8), respectively. Therefore, high hydrophobic content and low net charge, irrespective of peptide length, does not translate necessarily to high hemolytic activity. Minimum inhibition concentration (MIC) assays have shown that AMPs and toxins have a preference or selective affinity for bacterial membranes. A clear difference between eukaryotic and prokaryotic lipid membranes is the amount of anionic lipid.20 Therefore, the net charge of the cationic peptides may explain the affinity for a particular lipid membrane composition. Melittin, which carries a + 5 net charge, has lower MIC against both Escherichia coli and S. aureus compared with maculatin, which carries only a + 1 charge.8,21 Interestingly, the difference between these cationic peptides is not as marked for the highly negatively charged Gram-positive S. aureus compared with the less charged Gram-negative E. coli. However, the short AMP aurein, which has the same charge as maculatin, displayed greater MIC for both bacterial types, contradicting a direct relationship between peptide net charge and bactericidal activity, and there are many examples of anionic peptides with membrane-disrupting activity.22 Most cytolytic peptides transition from a random coil structure in aqueous environments to either an α-helix or a β-sheet structure in the presence of lipid membranes. The conformational change promotes a rigid peptide backbone (N− Cα−CO) that is stabilized by hydrogen bonds between the N−

Australian scorpion venoms. These peptides, in contrast to cell penetrating peptides (CPPs), are naturally designed to lyse eukaryotic cells and, therefore, are often anticipated to show a greater affinity for neutral membranes.

2. MEMBRANE DISRUPTION BY ANTIMICROBIAL PEPTIDES AND TOXINS AMPs and toxins have a wide spectrum of lytic activity but target lipid membranes for which they have higher affinity than the aqueous environment. Their adsorption onto membranes is driven by electrostatic and hydrophobic interactions and induces structural changes in both the peptide and the lipid membrane (Figure 1). Four AMPs found on the skin of Australian tree frogs1,8 are compared: aurein 1.2, citropin 1.1, maculatin 1.1, and caerin 1.1 are 13, 17, 21, and 25 amino-acid long peptides, respectively, with C-amidated termini (sequences are shown in Table 1) and carry a net positive charge. Their NMR solution structures were obtained in dodecylphosphocholine (DPC) micelles9−12 and are presented in Figure 2. Toxins from venoms are discussed to compare the effects of peptide net charge, hydrophobicity and length: melittin, the primary component of bee venom, is 26 amino acids long and has a net charge of +5, cupiennin 1a, found in Cupiennius salei spider venom, is 35 amino acid long with a +8 net charge, and UyCT5, isolated from the Australian scorpion Urodacus yaschenkoi venom, is 13 amino acids long with a +2 net charge. 2.1. Peptide Hydrophobicity and Net Charge versus Activity

Membrane-disrupting peptides are purported to have a greater content of hydrophobic amino acids (>50%), so as to insert into the acyl chain region of phospholipid bilayers. Indeed, the AMPs and toxins presented in Table 1 have hydrophobicity values from 51% to 68%. Surprisingly, these peptides are highly soluble and adopt a predominantly random coil conformation in buffered solutions as reported by CD experiments.13−16 Since red blood cell membranes have mainly neutral lipids, the hemolytic concentration of these peptides can be used as a C

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negatively charged vesicles, but if both are present, the AMP has to first saturate the latter before targeting the neutral vesicles (Figure 3).28 Therefore, assessing the activity of

H (donor) and CO groups (acceptor). The rigid backbone propensity may be responsible for modulation of the potency to disrupt the membrane bilayer organization, because peptides with a stable backbone are more likely to maintain their structure within the fluid membrane core and disrupt the surface tension and create membrane defects that ultimately break down the bilayer integrity. Moreover, the extent of the peptide secondary structure may also affect the backbone rigidity and thereby its ability to get into membranes and disrupt the bilayer integrity. As shown in Figure 2, aurein and citropin display a rather straight helical secondary structure, while maculatin and caerin are bent, due to the proline residues and highly curved DPC micelle environment used in the NMR studies.9−12 CD spectroscopy reports the helical content of aurein and UyCT5, two 13 residue peptides with similar charge, to be 74% and 55%,13,15 respectively, in the presence of neutral vesicles, with similar values of 70% and 65%, respectively, in the presence of anionic vesicles. The hemolytic concentration, however, for aurein is over 68 μM8 and that for UyCT5 14 μM.15 The toxin UyCT5 also exhibits significantly greater antimicrobial activity than the AMP aurein, which indicates that peptide length, charge, and hydrophobicity criteria are insufficient to explain the difference in membrane-disrupting activity between the two peptides. At three amino acids longer, the AMP citropin, which caries a + 2 charge, has slightly greater helical content in anionic sodium dodecyl sulfate micelles (78%) than UyCT5 and aurein. Citropin is weakly hemolytic (>62 μM)8 and has significant antimicrobial activity against S. aureus but not E. coli.23 Thus, in summary, predicting the hemolytic and bactericidal activities based on the hydrophobicity, charge, and secondary structure of AMPs and toxins remains elusive.

Figure 3. Single versus competitive dye release assay upon incubation of large unilamellar vesicles (LUV, 100 nm) with increasing amount of maculatin. Single lipid environment (open symbols) was made with 50 μM dye-encapsulated and 50 μM dye-free LUV of the same lipid composition: POPC (open squares) and POPG/TOCL (open circles). Competitive lipid environments (filled symbols) were prepared by mixing 50 μM POPC LUV and 50 μM POPG/TOCL LUV: calcein dye in POPC (closed squares) or POPG/TOCL (closed circles). Reproduced with permission from ref 28. Copyright 2014 Springer.

membrane-active peptides would be of greater benefit if performed in a competitive membrane environment rather than with a single type of vesicle present. Unfortunately, such assays are difficult to achieve with live cells (e.g., red blood cells versus bacteria) but can be easily performed with vesicles as model membranes. The membrane fluidity is also a key parameter for modulating the activity of cytolytic peptides. Cholesterol and sterols in general enhance the lipid packing and the order within cell membranes. Interestingly, the binding of AMP and insertion in cholesterol-rich membranes is often decreased, while toxins have shown higher affinity for a cholesterol-rich environment. Correspondingly, maculatin binds poorly to cholesterol-rich membranes, while melittin is extremely active in such an environment. Competitive assays were used to show that δ-lysin, a lytic peptide produced by S. aureus, preferentially binds to mammalian cell membranes, which are enriched in SM, cholesterol, and unsaturated PC.29 The lipid phase, such as gel versus fluid lamellar, can also promote differences in peptide−membrane interactions. As observed from 2H NMR spectra (Figure 4), aurein and caerin have a mild effect on the lipid packing in the fluid phase of deuterated DMPC bilayers, but in the gel phase, aurein induces a single isotropic population, while caerin did not significantly disturb the lipid organization. Interestingly, based on the ability to actively repair cell membrane damage, maculatin has a similar lytic mechanism for both E. coli and S. aureus, despite their different membrane structure and composition.17 Indeed, maculatin disrupted the lipid membrane structure of E. coli and S. aureus with a similar mode of action, although inhibition of growth occurred at much lower concentrations in Gram-positive bacteria. The former are rich in zwitterionic PE lipids, which also tend to be more

2.2. AMP Mechanism and Lipid Environment

Because membrane-active peptides target cell membranes, the lipid membrane architecture and properties need to be considered. In the case of eukaryotes and prokaryotes, membranes differ significantly in composition and organization. Eukaryotic cells are compartmentalized with organelles, such as the nucleus, mitochondrion, and the endoplasmic reticulum, which have distinct membranes. Each organelle has a different lipid composition but is mainly composed of unsaturated phosphatidylcholine (PC) with a small amount of anionic lipids, such as phosphatidylserine (PS) or cardiolipin (CL) for mitochondria, which are mainly located in the inner leaflet of the membrane.24 A significant amount of cholesterol is also found, for example, up to 40% in red blood cell membranes.25 Gram-negative bacteria have an inner and outer membrane. The outer leaflet of the outer membrane is mainly composed of lipopolysaccharides (LPS), while the inner leaflet of the outer membrane and the inner membrane are rich in saturated cyclopropane phosphatidylethanolamine (PE) and have a significant amount of anionic phosphatidylglycerol (PG) and CL.24 On the other hand, Gram-positive bacteria have only one lipid membrane comprising mainly anionic PG and CL lipids.26 Little is known about sterols in bacteria, but recent studies show that hopanoids can play a significant role in bacterial membrane modulation.27 Interestingly, melittin and maculatin have the ability to interact with neutral and negatively charged vesicles, although the affinity is often greater for the latter. However, affinity does not translate necessarily to activity. For instance, maculatin is about 10-fold more potent to disrupt neutral compared with D

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Figure 4. Deuterium NMR spectra of d54-DMPC at (a) 30 °C and (d) 15 °C, d54-DMPC + aurein 1.2 (10:1) at (b) 30 °C and (e) 15 °C, and d54DMPC + caerin 1.1 (10:1) at (c) 30 °C and (f) 15 °C. Reproduced with permission from ref 13. Copyright 2013 Elsevier.

Figure 5. Lipid composition regulates conformation and insertion of maculatin 1.1. The α-helical content of the peptide is highest when the helical length of the AMP matches the hydrocarbon thickness (DMPC). Reproduced with permission from ref 16. Copyright 2011 Elsevier.

attributed to the ability of the peptide to span the hydrophobic core of the bilayer, for example, short peptides (∼8−15 residues), such as the AMP aurein, act as a detergent (micellization),30 while longer peptides, such as maculatin (21 residues), form transbilayer pores.31 The length dependent activity of pore forming AMPs32,33 can be related to the hydrocarbon thickness of the bilayer that matches the length of the helical peptide (Figure 5). In an experiment using dyes of different size that were coencapsulated, maculatin was able to release molecules as large as 40 kDa (Figure 6), and since small vesicles were not detected upon the peptide incubation, a pore mechanism has been proposed.31 Highest dye release was seen with vesicles of anionic phospholipids with unsaturated chains.16,28,31 The peptide activity is modulated by both the thickness and fluidity of the bilayer, and the AMPs must self-associate in order to form pores.

ordered due to the smaller headgroup, and the latter are high in anionic PG and CL. In addition there is diversity in the lipid acyl chains, which would modulate membrane properties as bacteria adjust their membrane lipid composition by modifying the types of fatty acids. 2.3. Structure−Function Relationship

Since a single peptide is unable to form a pore in a lipid membrane, the tendency to self-associate is primordial to peptide activity, which is consequently concentration-dependent. On encountering a membrane, peptides may either aggregate on the surface or, once they reach a critical concentration, insert into the membrane. Electrostatic interactions often “lodge” the peptides at a superficial depth, while hydrophobic interactions drive the peptides into the hydrophobic core of the bilayer. The two conduits generally trigger a different lytic mechanism. The different mechanism of lipid bilayer disruption, pore formation versus micellization, is E

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CPPs.39−43 However, the molecular mechanism of membrane translocation is still to be elucidated since as yet no structure− function relationship has been established. A specific secondary structure is not necessary for CPPs to translocate across lipid bilayers because peptides adopting random coil, β-sheet, and α-helical structures traverse membranes. However, CPPs that adopt a β-sheet or an αhelix require a high membrane content of negatively charged lipids since they remain unstructured in aqueous solutions and in the presence of vesicles made of neutral lipids such as PC or with a low content of anionic lipids. CPPs with random coil structures, in general, have a shorter primary sequence (+8 net charge) than those that adopt a secondary structure. However, many CPPs transition from a random coil to defined secondary structure following interaction with lipids or polysaccharides, and some AMPs have been proposed to act via a similar mechanism to the CPPs.

Figure 6. Effect of maculatin concentration on the release of rhodamine−dextran 40 kDa (RD-40, solid bars) and fluorescein− dextran 4 kDa (FD-4, cross-hatched bars) from POPG/TOCL LUV. Reproduced with permission from ref 31. Copyright 2013 American Society for Microbiology.

3.1. Proline-Rich Antimicrobial Peptides

One such class of AMPs that translocate rather than insert into membranes is the proline-rich antimicrobial peptides (PrAMPs).44−46 These proline-rich AMPs are a group of cationic host defense peptides often found in insects and characterized by a high content of proline residues together with arginine (or lysine) residues in repeated motifs. Rather than a lytic mechanism, their mode of action involves cellpenetration whereby they subsequently inhibit intracellular targets. The PrAMPs, apidaecins and oncocins, bind to the 70S ribosome and the chaperone DnaK to inhibit protein biosynthesis in Gram-negative bacteria. Not only are PrAMPs able to modulate the immune system via cytokine activity or angiogenesis, but they also possess properties of CPPs and are able to penetrate cell membranes and cross the blood−brain barrier. PrAMPs show a variety of modes of actions, including a mechanism shift at higher concentrations, nonlytic mechanisms, and different intracellular targets and binding to the lipopolysaccharide on the outer membrane of bacteria.47 Consequently PrAMPs have potential as anti-infective lead compounds and also as CPPs for drug delivery into both bacterial and eukaryotic cells. Hence, PrAMPs have been studied extensively to determine their specific modes of action. A de novo designed branched dimeric PrAMP, A3-APO (HChex-(RPDKPRPYLPRPRPPRPVR)2-Dab, with Chex = Cyclohexane-carboxylic acid and Dab = 1,3-diamino-propionic acid), is highly effective against a variety of in vivo bacterial infections. We examined the mode of action of this PrAMP by studying the effect of multimerization on the membrane interaction with the Gram-negative bacteria E. coli using the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the monomer showed no membrane activity, while the tetramer was 30-fold more membrane disruptive than the dimer. Flow cytometry and highresolution fluorescent microscopy showed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from nonlytic/membrane hyperpolarization to membrane disruption and depolarization.44 Thus, the membrane interaction and permeability of the PrAMP Chex-Arg20 was enhanced by oligomerization. Therefore, PrAMP oligomers may have a role as potential novel carriers or drug delivery agents and may lead to the development of more potent AMPs against bacterial infection.

At present, very little is known on the self-association of AMPs, which are often described as forming transient structures and toroidal pores that are not selective to a specific metabolite or ion.34−36 Although the lipid composition has often been shown to modulate the activity of lytic peptides, maculatin is able to trigger very similar membrane damage in neutral and charged phospholipid bilayers. However, the AMP preferentially targets the latter (Figure 3), which indicates that lipid environment may simply modulate the binding but not the activity of these peptides.17,28 Moreover, peptides can act synergistically,37 as has been observed for two AMPs, PGLa and magainin 2, which was isolated from the African frog Xenopus laevis.38 Both peptides form α-helices when bound to phospholipid bilayers. The peptides on their own are either in surface orientation or tilted in the membrane, but when the peptides are mixed in a 1:1 molar ratio, PGLa changes to the fully inserted state, whereas magainin stays on the membrane surface. Thus, formation of transmembrane pores, which would not spontaneously form by either peptide alone, appears to be the basis for the synergy. Similarly a CPP could be combined with an AMP so as to produce synergy in the interaction of these membrane-active peptides with lipid bilayers.

3. PEPTIDE TRANSLOCATION THROUGH LIPID MEMBRANES Membranes present a barrier to hydrophilic molecules that interact with intracellular targets. Peptides with cell-penetrating activity, which are able to cross cell membranes rather than insert, are used to translocate cargo into cells. These cellpenetrating peptides (CPPs) share many features with AMPs and may exhibit antimicrobial activity. CPPs have a common feature: they possess a high number of lysines or arginines or both and, therefore, are highly positively charged. Their interaction with lipid membranes thus is assumed to be driven by electrostatic interactions with negatively charged lipids, such as PS and PG. Based on the primary sequence of natural CPPs (e.g., TAT, penetratin, PVec, M918), several peptides (e.g., MAP, EB1, Arg9, CADY) have been designed in order to identify the important features and improve the activity of F

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Figure 7. Solid-state NMR shows effect of kalata B1 (kB1) on phospholipid membranes and the role of PE: (A) Static and (B) magic angle spinning 31 P NMR spectra of POPC/DOPE (1:1) bilayers. Insets show deconvolution of the MAS spectra using Lorentzian functions and their sum (dashed lines). (C) Pake and (D) dePaked 2H NMR spectra of POPC/d31-POPC/DOPE (1:1:2). Spectra without (black) and with (gray) kB1 in multilamellar vesicles at peptide/lipid 1:10. Reproduced with permission from ref 49. Copyright 2015 Elsevier.

possibility could be that CPPs are capable of acting as a monomer and hence do not stay in the membrane. AMPs may self-assemble to form a pore or an aggregate that remains in the bilayer, acting somewhat like a pore-forming protein or a peptide−lipid complex that destabilizes the membrane. However, by combining properties of both translocation and membrane affinity, AMPs are able to get into bacterial membranes and lead to the destruction of the cell. As well as combining the properties of CPPs and AMPs to enhance antimicrobial activity, a synergistic approach could be tried by pairing AMPs, as in the case of PGLa and magainin discussed earlier. In addition, future studies of the structure−function relationship of AMPs could be conducted in-cell using NMR techniques,50 as well as focusing more on peptide−peptide interactions and their interactions and assembly within the membrane.

By combining both CPP and AMP properties, more effective therapeutics against multidrug resistant microbes may be designed. 3.2. Membrane Lipid Modulates Activity of Kalata B1, a Cyclic CPP

Backbone cyclization is often proposed as a method to confer stability on peptides. Cyclotides are disulfide-rich head-to-tail cyclized peptides from plants. They combine the stability of disulfide-rich peptides with the intracellular accessibility of CPPs, hence giving them outstanding potential as drug scaffolds with an ability to inhibit intracellular protein−protein interactions. The interaction of the prototypic cyclotide kalata B1 (kB1) with model mammalian membranes was studied by solid-state 31P and 2H NMR (Figure 7). In the presence of kB1, a significant reduction in the overall chemical shift anisotropy and the formation of isotropic peaks (centered at ∼0 ppm) was observed in the 31P NMR spectra (Figure 7A,B). 2H NMR, however, showed that the perdeuterated acyl chains were not significantly disturbed (Figure 7C,D). There was no evidence of hexagonal-phase formation, which is often an intermediate step in the internalization of other CPPs, such as TAT.48 Kalata B1 can enter cells via both endocytosis and direct membrane translocation.49 Using NMR and other biophysical methods, kB1 was shown to target specifically phosphatidylethanolamine (PE) phospholipids at the cell surface and induce membrane curvature to initiate the cell entry. This approach could be employed to deliver drugs into cells and, in particular, Gramnegative bacteria, which have a high proportion of PE phospholipids. By combining the properties of both CPPs and AMPs, this cyclic peptide may inspire design and discovery of more effective antimicrobials.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Marc-Antoine Sani (b. 1979) obtained an MS. in physical chemistry in 2004 from the University of Bordeaux (France) and a Ph.D. in biophysics in 2009 cojointly from the University of Umea (Sweden) and the University of Bordeaux. He is a senior postdoctoral fellow in the School of Chemistry at the University of Melbourne. His research interests are the study of membrane-active peptide and protein interactions with complex lipid architectures and development of solidstate NMR techniques to extract intricate information from these heterogeneous systems.

4. CONCLUSIONS Clearly membrane-active AMPs do not act alone, and more than one peptide is needed to form a pore. However, the details of how AMPs self-assemble and form a pore remain unclear. In fact, whether pores are formed or AMPs simply destabilize the bilayer by making aggregates is not clear, and both mechanisms may exist depending on the peptide, its concentration, and the membrane. AMPs share properties with CPPs and the differences between a CPP and an AMP are not clear. One

Frances Separovic obtained a B.A. in mathematics and physics in 1986 from Macquarie University (Australia) and a Ph.D. in physics in 1992 from the University of New South Wales (Australia) while studying part-time and working full-time at the CSIRO in Sydney (Australia). Following a postdoctoral fellowship in biophysics in 1994−1995 at the NIH (Bethesda, MD, USA), Frances was appointed an associate professor and reader in Chemistry in 1996 at the University of Melbourne (Australia). Frances became the first woman G

DOI: 10.1021/acs.accounts.6b00074 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

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full professor of chemistry in the state of Victoria in 2005 and Head, School of Chemistry, in 2010−2015. Her research is focused on the study of membrane-active peptides and toxins in situ and biological solid-state NMR spectroscopy.

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DOI: 10.1021/acs.accounts.6b00074 Acc. Chem. Res. XXXX, XXX, XXX−XXX