membrane interacting peptides: from killers to helpers

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620 Current Protein and Peptide Science, 2012, 13, 620-631

Membrane Interacting Peptides: From Killers to Helpers

Erick J. Dufourc1,*, Sébastien Buchoux2, Jeannot Toupé1, Marc-Antoine Sani3,Frantz Jean-François4, Lucie Khemtémourian5, Axelle Grélard1, Cécile Loudet-Courrèges6,Michel Laguerre1, Juan Elezgaray1, Bernard Desbat1 and Benoit Odaert1

1Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN) UMR5248, Université Bordeaux, CNRS, Insti-tut Polytechnique Bordeaux, Pessac, France; 2Enzyme & Cell Engineering (GEC) UMR6022, Université Picardie Jules Verne,CNRS, Amiens, France; 3Chemistry Department, University of Melbourne, Parkville, Vic 3052, Australia; 4Dept of Biochemistry & NHMFL, Florida state University, Tallahassee, FL 32310, USA; 5Laboratoire de Biomolécules, UMR7203 ENS,UPMC,CNRS, 75252 Paris Cedex 5, France; 6IECB, UMS3033, Université Bordeaux,CNRS, 33600 Pessac, France

Abstract: Membrane interacting peptides are reviewed in terms of structure and mode of action on lipid membranes. Helical, �-stranded, peptides containing both helices and strands, cyclic, lipopeptides and short linear peptides are seen to considerably modulate membrane function. Among peptides that lead to membrane alteration or permeation, antimicrobial peptides play an important role and some of them may be foreseen as potential new antibiotics. Alternatively, peptides that do not destroy the membrane are also very important in modulating the structure and dynamics of the lipid bilayer and play important roles in membrane protein functions. Peptide lipid complexes are shown to be very variable in structure and dynamics: “carpet”, “barrel stave”, toroid and disordered pores, electrostatic wedge and molecular electroporation models are discussed. Their assembly is reviewed in terms of electric, amphipathic and dynamic properties of both lipids and peptides.

Keywords: Helical-, beta-sheet-, cyclic-, linear-peptides, membrane leakage, membrane reinforcement, membrane crossing, NMR, IR, CD, X-rays, electron microscopy, molecular dynamics.

INTRODUCTION: MEMBRANE PEPTIDES, 3D STRUCTURES AND ACTION

Membrane interacting peptides play essential roles in cell fate. They may kill cells or help them in adapting to external constraints. As an example, the increasing resistance of bac-teria to conventional antibiotics has pushed for the develop-ment of new modes of treatment and over the past few years, antimicrobial peptides (AP) have been presented as a poten-tial solution to this problem [1]. Whereas classical antibiotics act specifically on biosynthetic pathways, antimicrobial pep-tides may directly destabilize the lipid membrane and consti-tute a promising alternative strategy for fighting microorgan-ism action. There is also a second class of peptides that are not antimicrobial but are rather targeted to the membrane interior as part of membrane proteins or signalling molecules and participate to membrane stability and function. Whereas some of the latter may associate as bundles to form mem-brane protein pores, the AP are rather small and were first identified in the hemolymph of insects and in the secretion of immune cells. Insects are indeed almost the only entities that produce such peptides as part of a systemic response induced by microorganisms [2-5]. For mammals, these peptides are part of the innate immune system that constitutes a first host defence line by controlling natural flora. By 2011, more than

*Address correspondence to this author at the CBMN, University Bordeaux-CNRS-IPB, Allée Geoffroy St Hilaire, 33600 Pessac, France; Tel: +33540006818; E-mail: [email protected]

1700 different antimicrobial peptides had been identified (see http://aps.unmc.edu/AP/main.php). In most cases, the peptide mode of action appears to be by direct lysis of the pathogenic cell membrane. They may be antimicrobial or/and haemolytic with their best activity in the sub �Mscale. Considering both AP and membrane peptides, one may distinguish several 3D structures: helices, beta sheets, pep-tides containing both helices and beta-sheets, cyclic, and short linear peptides Fig. (1). Their length ranges from 5 to 50 amino acids and they can bear positively (Arg, Lys, His) or negatively (Asp, Glu) charged residues that may condition their interaction with zwitterionic mammalian membranes or negatively charged microbial membranes. As shown in (Ta-ble 1), they may also contain amino acids showing a marked hydrophilic (Asn, Gln, Pro) or lipophilic (Phe, Trp, Tyr, Leu, Ile, Met) character that will drive their location into mem-branes. Combining the electric and amphipathic properties of amino acids, peptides may show charges and hydro-philic/phobic regions that will be located at peptide extremi-ties (transmembrane peptides) or along peptides faces (an-timicrobial peptides), Fig. (1). The 3D distribution of electric and amphipathic character will condition their action. One may distinguish two main families of membrane active peptides, those that act on the lipid part and those that go directly to membrane proteins or receptors and trigger or impede a function. As we will see

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Membrane Interacting Peptides: From Killers to Helpers Current Protein and Peptide Science, 2012, Vol. 13, No. 7 621

later there is no clear cut difference between the two families and there may be peptides that need also to pass through the lipid part to interact with receptors. In the present review we will discuss peptides that interact with lipid membranes. Re-views on peptides that directly bind to membrane proteins may be found elsewhere [6-8]. Among peptides that interact with membrane lipids one may distinguish those that are embedded in the lipid bilayer and participate in membrane function as structural peptides and those that may lead to membrane disruption (“killer” peptides) or membrane per-meation (pore forming peptides). Again a sorting into well-defined classes of action may appear subtle as it has been reported that peptides may disrupt membranes at high con-centration and permeate them at low doses and there are also examples of membrane pores made by helix bundles or beta barrels. Because it is difficult to sort peptides associated to func-tion, we will describe in this review membrane action ac-cording to peptide 3D structure upon lipid binding. The structural viewpoint has the advantage of simplicity and is also linked to the three main physio-chemical forces that

condition interaction, that is to say, hydrophobic/philic, elec-trostatic and hydrogen bonding. FORCES CONDITIONING INTERACTION WITH MEMBRANES AND METHODS FOR STUDY

A lipid membrane is by essence an anisotropic medium that globally offers a hydrophobic core (hydrocarbon chains) and a hydrophilic interface starting from the glycerol back-bone or the sphingosine skeleton for glycerol- or sphingo-based lipids, respectively, and ending at the last head group atom in the water medium. When a peptide has to go through or must be embedded into a membrane it will encounter, coming from the cell exterior, a first hydrophilic/charged layer of 10-15 Å, then a hydrophobic layer of 20-30 Å and a second hydrophilic/charged layer of 10-15 Å. The length of the respective layers may be variable and there may not be symmetry in charges and hydrophilic thicknesses according to the intrinsic asymmetric character of natural bilayer mem-branes. In addition to peptide spatial partitioning into amphipa-thic regions, membrane dynamics has also to be taken into

Fig. (1). Tri-dimensional structures of helical, �-stranded, CS��, cyclic and short linear peptides. The representation includes the ribbon representation when �-helices and �-strands are present. The colour code represents hydrophobicity (seen in Table 1): blue = hydrophobic, red = hydrophilic. Tyrosine kinase receptor NeuTM35, PDB: 1IIJ (56); Protegrin, PDB: 1PG1 (90); �-Hemolysin, PDB: 2KAM (45); Cathelicidin, PDB: 1LXE(91); Surfactin, adapted (hydrocarbon chain added) from PDB: 2NPV (92); Menk, methionine encephalin, structure in membranes from (25);

622 Current Protein and Peptide Science, 2012, Vol. 13, No. 7 Dufourc et al.

Table 1. Hydrophilic & Lipophilic Amino Acid Properties

Amino Acid Side Chain Hydrophobicitya Free Energy (kcal/mol)b Hydrophobicityc

pH 8 pH 2

Ala A Alanine CH3 1,80 -0,17 0.11

Arg R Arginine (CH2)2NC(NH2)2 -4,50 -0,81 2.58

Asn N Asparagine CH2CONH2 -3,50 -0,42 2.05

Asp D Aspartic Acid CH2COOH -3,50 -1,23 0,07 3.49

Cys C Cysteine CH2SH 2,50 0,24 -0.13

Gln Q Glutamine (CH2)2CONH2 -3,50 -0,58 2.36

Glu E Glutamic acid (CH2)2COOH -3,50 -2,02 0,01 2.68

Gly G Glycine H -0,40 -0,01 0.74

His H Histidine CH2(C3N2H3)cyc -3,20 -0,17 -0,96 2.06

Ile I Isoleucine CHCH3CH2CH3 4,50 0,31 -0.60

Leu L Leucine CH2CH(CH3)2 3,80 0,56 -0.55

Lys K Lysine (CH2)4NH2 -3,90 -0,99 2.71

Met M Methionine (CH2)2SCH3 1,90 0,23 -0.10

Phe F Phenylalanine CH2(C6H5)cyc 2,80 1,13 -0.32

Pro P Proline (CH2)2cyc -1,60 -0,45 2.23

Ser S Serine CH2OH -0,80 -0,13 0.84

Thr T Threonine CHOHCH3 -0,70 -0,14 0.52

Trp W Tryptophan CH2(C8NH6)cyc -0,90 1,85 0.30

Tyr Y Tyrosine CH2(C6H4)cycOH -1,30 0,94 0.68

Val V Valine CH(CH3)2 4,20 -0,07 -0.31

aA simple method for displaying the hydropathic character of a protein [87]. The more positive the numbers in column, the greater the hydrophobic character. bExperimentally determined hydrophobicity scale for proteins at membrane interfaces [88]. Positive values indicate partition of short peptides containing the aminoacid of interest into the neutral (zwitterionic) phospholipid membrane phase, negative values, partition into the water phase, accuracy 10%. cRecognition of transmembrane helices by the endoplasmic reticulum translocon [89]. Using a set of designed polypeptide segments, it has been determined the basic features of this 'biological' hydrophobicity scale. In this scale (unlike the others), more negative values reflect greater hydrophobicity

account. The most rigid part of the membrane is located at the glycerol or sphingosine backbone level where the fatty acid chains attach [9]. The head group facing the water is very mobile towards its end and the hydrocarbon chains offer also a great mobility at the membrane centre. For chain posi-tions near the glycerol backbone a plateau of quite elevated rigidity is detected. Membrane dynamics is also complex as lateral phase separation may occur. There is increasing evi-dence that lipid domains of different dynamics are present in membranes and that membrane peptides and proteins parti-tion differently according to the lateral and trans-membrane dynamics. Cholesterol and sphingolipids are known to play crucial roles in the formation of these so-called “rafts” [10]. Peptide interaction with membrane lipid layers may be followed by several techniques. There are techniques that report on morphological changes as induced by peptides such as Electron Microscopy, AFM, Brewster microscopy, fluorescence imaging [11-15]. These techniques have differ-ent advantages and drawbacks; whereas Electron microscopy

and AFM have a great spatial resolution, the sample prepara-tion may generate artefacts. On the other hand classical fluo-rescence has a great sensitivity but uses dyes that may per-turb systems and has a limited resolution of ca. 30 nm. Brewster microscopy does not use markers but is most often reduced to the tenth of micrometre scale. There are other techniques that report on global changes: calorimetry allows finding whether interacting peptides perturb the thermody-namic properties of the membrane [16, 17] and solid-state NMR and X-rays report on phase changes [16, 18, 19]. Techniques such as EPR, IR, X-rays and NMR report inter-action at the atomic level and are capable of describing both peptide and lipid structure and dynamics. Secondary peptide structure upon binding can easily be obtained from IR (using ATR or PMIRRAS methods), or circular dichroism [20-22]. Such techniques can also provide information about peptide orientation [23, 24]. NMR will be the only technique capable of yielding both the topology and the three dimensional structure of peptides in membranes by combining magic an-


gle sample spinning and oriented systems techniques [25, 26]. Extensive reviews on using oriented samples can be found in [27, 28]. Computational methods have been widely used for study-ing peptide membrane interactions. The first approach has been the mean field method that employs an empirical en-ergy function (hydrophobicity potential) where the lipid bi-layer is represented by a slab of low dielectric constant and water by other slabs of high dielectric constants. Insertion of helical peptides have been predicted with success (for a re-view see [29]) but the approach is however limited to the fact that peptide-peptide interactions are hard to take into account and the gradients of mobility and dielectric constant across the bilayer are not accounted for. With the increase of com-puter power all-atom molecular dynamic (MD) simulations have been and are still widely used. They are based on New-ton equations of motion (force calculation and hence accel-eration of each atom) and yield the coordinates of all atoms in a system as a function of time [30]. A very comprehensive review on MD of lipid-peptide interactions, yielding nice representations of �-helix topologies and structures when inserted or applied onto bilayers can be found in [31]. The main problem resides in defining the correct initial condi-tions, because intrinsic time limits are bound to such an ap-proach. The time problem may be circumvented at loss of resolution in the Coarse Grain (CG) and Dissipative Particle Dynamics (DPD) approaches. The global idea is to replace a large cluster of atoms by a single interaction site. In a recent review Marrink and co-workers mention that such methods allow simulation at the micrometre and millisecond scales. Nice applications to calculation of peptide-induced mem-brane poration can be found in [32].

MEMBRANE ACTIVE PEPTIDES

Helices

Peptides that adopt a �-helical structure are by far the most studied. For reviews see [1, 33-39]. Those that damage the membrane by direct lysis, i.e., entire membrane restruc-turation, or induce membrane leaks without breaking the cell membrane originate from insect toxins, microorganisms, vertebrates as well as bacteria. Melittin, �-Hemolysin, Ma-gainin, Bombolitin, Mastoparan, Crabolin, Alamethicin, Trichorzianin A, Cecropin B, Pardaxin are some specimens of this family of amphipathic helices [40]. They share com-mon structural properties: they are of 13-35 amino acid length, mostly basic (1 to 6 positive charges) and show a helical amphipathicity, that is to say, their lipophilic amino acids appear all along the helix on one face, the charged and hydrophilic ones on the other face (e.g., Fig. (1), �-Hemolysin). The helix may be regular or bent if a Pro resi-due sits in the middle of the sequence like Melittin or Alame-thicin. Their mode of action has been extensively studied and one often refers to the “carpet” or “barrel stave” mechanisms to describe their action on membranes [37]. The first mecha-nism that conducts to membrane solubilisation is akin to detergent-like action [39, 41] and has been extensively dem-onstrated for Melittin, a bee-venom toxin, using Electron Microscopy, NMR, dynamic light scattering and many other structural techniques [42, 43]. It is proposed that peptides cover the cell surface until a threshold concentration is

reached that leads to the formation of membrane patches in which the lipids form toroid aggregates stabilized by the amphipathic peptides. Dramatic membrane disruption thus occurs, leading to cell death. The new structure that is formed by the lipid-Melittin assemblies resembles to the apolipoprotein lipid complexes where the amphipathic pep-tides sit in the outside of a small disc of 20-40 nm diameter, offering their lipophilic helix face in contact with lipid chains, Fig. (2). Interestingly these discs are preferentially made with highly ordered lipids or lipid mixtures containing cholesterol. The model described in Fig. (2) for Melittin ap-pears to hold true for Magainin 2 as well [44]. It is also ex-pected that higher doses of peptides will lead to mixed mi-celles with a barely defined structure.

Fig. (2). Schematic representation of the end process in the “carpet” model for Melittin action on large lipid multi-layered vesicles. Upon sufficient surface concentration, peptides cut the membrane into small discs which dimensions have been determined by elec-tron microscopy, light scattering and NMR (42). In the discs, phos-pholipids and cholesterol are surrounded by Melittin either with its helical axis parallel or perpendicular to the bilayer plane (43).

μm


The “barrel stave” mechanism aims at describing mem-brane permeation through pores where helical peptides such as �-Hemolysin, aggregate as helix bundles in the membrane core with their hydrophobic face in contact with lipid chains and the hydrophilic one making the interior of the pore in a way similar to some membrane protein channels [38, 45, 46]. Here, the minimal inhibitory concentration required for dis-sipating the transmembrane potential should be far below the �M concentration, a fact only observed for a few peptides, such as Alamethicin and Pardaxin [47, 48]. Although con-vincing electrophysiology measurements of transmembrane currents, and fluorescence leakages of internally trapped dyes have been reported there is, so far, no structural evi-dence of such a structure by neither high resolution X-rays nor NMR. It must however be mentioned that pores have been reconstructed to low-resolution using small angle X-ray scattering or anomalous diffraction [49, 50]. Natural antimi-crobial peptides have been modelled by simplifying the se-quence using only Leu or Lys residues [33, 34, 51, 52]. Very powerful antimicrobial peptides were thus built using an average of 18 alternating Leu/Lys residues that were able to stabilize as helices showing secondary amphipathicity, i.e.,clustering of hydrophilic/charged residues on one face of the helix, hydrophobic residues on the other face. Among helical peptides leading to membrane permea-tion, the gramicidin channel deserves a special mention. Such an unusual 15 amino acid peptide is produced by the soil bacteria Bacillus brevis during its sporulation phase and shows killing activity against Gram-positive bacteria. It is made of L and D amino acids and mostly presents a �-helical structure in membranes where it forms a tail-tail dimer lead-ing to a hydrophilic internal transmembrane pore specific for the transport of monovalent cations across membranes [53, 54]. Of interest such a structure does not lead to membrane disruption as was discussed above for the “carpet” mecha-nism of �-helices, it only generates cation leakage. The mechanism is therefore more related to that of the “barrel stave” model except that only two monomers are required to make the pore. There are helical peptides that do not show such an activ-ity, nor disrupt the membrane. They contribute to the bilayer stability, in a way very similar to membrane proteins. In general they have a clustering of hydrophobic amino acids that corresponds to the membrane hydrophobic interior. The transmembrane sector of Neu/ErbB2 shows such a structure Fig. (1). It belongs to growth factor receptors of the tyrosine kinase family and link separately folded domains, one exter-nal and one cytosolic. The sequence of the membrane-spanning 35 amino acid peptide appears to be important for function. Indeed, a single point mutation in the proto-oncogene neu, resulting in a substitution of a valine residue for glutamic acid at position 664 within the transmembrane region, transforms it into an oncogene [55, 56]. The mutant receptor then has constitutive tyrosine kinase activity in the absence of ligand, apparently as a result of greatly enhanced receptor dimerization. The membrane spanning peptide is an ��-helix made of very hydrophobic residues. The �-helix is constituted by a series of 4 Valine residues and is believed helping for adaptation to the hydrophobic mem-brane mismatch by presenting a reserve for helix elasticity.

Killian, Mouritsen and co-workers have extensively re-viewed the concept of adequate matching between the length of the hydrophobic transmembrane peptide segment and the length of the lipid bilayer hydrophobic core [57-61]. Effects of mismatch on protein activity, stability, orientation, aggre-gation state, localization, and conformation have been ap-proached using model helical peptides of variable length, called WALP or KALP because they have a hydrophobic span made of Ala and Leu residues, anchored on each side of the lipid bilayer by Trp or Lys residues. Varying the bilayer thickness using C12 to C20 phospholipids afforded follow-ing the bilayer response. The authors proposed that both lip-ids and peptides adapt their length to compensate for unfa-vourable hydrophobic/philic interaction energy. When helix bundles are made through peptide aggregation or in a mem-brane protein the lipid in the vicinity of the bundle in-creases/decreases its length. In the case of single spanning peptides the peptide may adapt its length (vide supra,Neu/ErbB2) or topology [62, 63] and may play a role in the appearance of lateral membrane clusters, sometimes called “rafts” into which receptor may localize [10]. There are peptides that show an intense conformational plasticity, i.e., exhibiting random, �-stranded or helical struc-tures depending on the medium (water, lipids) they are fac-ing. The role of such plasticity is not well understood. It may be linked to the different roles peptides may play in living cells. Such a phenomenon has been encountered for instance in key events such as programmed cell death following the translocation of the apoptotic Bax protein from the cytosol towards mitochondria. Whereas anti-apoptotic proteins are originally anchored in the outer mitochondrial membrane, the main pro-apoptotic protein, Bax, is located in the cytosol as inactive. However, binding of an external stimulus to Bax in known to induce a conformational change and expose targeting/anchoring domains and trigger translocation to the mitochondria membrane. It has been proposed that the first helix localized at the N-terminus of Bax (Bax-�1) may act as an addressing sequence. Solid-state NMR, CD and ATR-IR spectroscopy have been used to elucidate this recognition process [13, 21]. Two potential target membranes have been studied: the outer mitochondrial membrane (OM) was mim-icked by neutral phospholipids PC and PE whereas mimics of mitochondrial contact sites (CS) were obtained by addi-tion of anionic cardiolipin, CL. It was found that Bax-�1induced pronounced perturbations in the lipid head group region only in presence of CL. Bax-�1 could not insert into CS membranes but at elevated concentrations it inserted into the hydrophobic core of cardiolipin-free membranes, where it adopted a �-sheet structure, Fig. (3) [64]. Further studies revealed that the CL-mediated electro-static locking of Bax-�1 at the CS membrane surface pro-motes intense conformational changes from �-strand to �-helix. Such a process appears to be necessary for inducing further conformational transition events in Bax that finally cause irreversible membrane permeation during the mito-chondrial apoptosis. The specific interaction between cardi-olipin and Bax-�1, stabilizing a helical conformation, would then be required for addressing Bax to the correct location in the mitochondria, and the presence of cardiolipin could act as a strong signalling event for Bax translocation.


Beta-Sheet Peptides

Although less common than helices, peptides that are already folded as �-sheets or that acquire the �-sheet struc-ture upon binding to membranes represent an important class of molecules. They originate from insects, frogs, crabs and mammals and are antimicrobial and fungicidal in the �Mrange. Very interestingly, and though very basic (4-8 posi-tive charges), they are weakly haemolytic, which makes them interesting candidates for bactericidal treatment [5]. They are in average shorter than �-helical peptides with lengths of 16-24 residues and unlike �-helices, �-strands are not stable as isolated secondary structures in solution unless the structure contains several cysteine bridges that lock the peptide into a �-sheet hairpin structure Fig. (1). Beta-sheet peptides may exist as parallel or antiparallel assemblies. Be-cause extensive reviews have been made on antimicrobial peptides showing a cysteine-stabilized �-sheet structure [1, 5, 65-67], we will concentrate on peptides that have diverse structure or none in solution and that acquire a �-strand to-pology upon interaction with membranes [68]. Antimicrobial peptides resulting from the enzymatic degradation of chro-mogranin A (CgA) and secreted during stress fall into this category [69, 70]. CgA is located in the secretory granules of most endocrine end neuroendocrine cells. It was recently discovered that a 15 amino acid peptide named Cateslytin (344RSMRLSFRARGYGFR358), displays antimicrobial ac-tivities in the �M range and has no detectable haemolytic action at concentrations of 100 mM. Recent work shows that it acts also on Plasmodium falciparum raising new hopes for the treatment of Malaria. Using a complete set of physical

techniques including solid and liquid state NMR, CD, ATR-IR, Electrophysiology and molecular modelling it has been shown that Cateslytin, which is unstructured in solution, is converted into antiparallel �-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic mem-branes, in a way similar to Alzheimer peptides [71]. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the Cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found Fig. (4). Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of �-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. It was also demonstrated that a single peptide is able to form a stable membrane pore of 1 nm diameter and 0.25 nS conductance as inferred both from molecular dynamics and electrophysiology measurements. The resulting structure does not resemble the “barrel-stave” or “carpet” models earlier discussed, but is a rather disor-dered pore promoted by molecular electroporation forces [26], i.e., the cationic peptide binding to the external side of the membrane induces a transmembrane electric field gener-ating a water pore followed by larger defects allowing pep-tide crossing through the membrane Fig. (4, insert). Of great interest, the specificity of Cateslytin to fungi membranes containing ergosterol has been demonstrated by a 2-step electronic interaction: attractive dipole-dipole between basic arginine residues and negatively charged lipid head groups, and attractive cation-� between arginine and the conjugated

Fig. (3). Schematic model of Bax-�1 interaction with two different mitochondrial membrane locations. Hydrophobic interactions occurring at the outer membrane, mainly composed of neutral lipids, PC and PE, induce partition between �-sheet inserted structures (especially at high concentration) and random coil conformations remaining in solution (orange colour). Contact site membranes, where native pores are stabi-lized (shown as an example here), are enriched by the anionic cardiolipin. The negatively charged surface locks the peptide onto the surface by electrostatic interactions and triggers �-helical secondary structure changes (green colour). From (64).


� electrons of the ergosterol fused-ring system [72, 73]. The complex leads to fluid/thinner membranes that laterally sepa-rate out from rigid/thicker membranes that are not bound by cateslytin. The intrinsic greater membrane fluidity of ergos-terol/acidic lipid components in fungi is shown to be one of the key factors for specific Cateslytin biological action.

�-stranded peptides are also involved in apoptosis. Se-quence analysis of the anti-apoptoticBcl-2 proteins reveals four short conserved domains (BH1 to BH4) whereas, for pro-apoptotic proteins such as Bax and Back, a similar over-all primary structure is found except for the BH4 domain (10DNREIVMKYIHYKLSQRGYEW30). The N-terminus do-main thus appears as a key factor for the regulative anti-apoptotic activity of Bcl-2 proteins at the mitochondrion level. Interactions of this peptide with membranes modelling the outer mitochondrion lipid composition have been fol-lowed by solid state NMR, differential scanning calorimetry, circular dichroism and IR-ATR methods [74, 75]. Coexis-tence of small sized fluid and rigid membrane domains over a large temperature range was found, the latter being stabi-

lized, in a cholesterol-like manner, by the presence of the BH4 peptide. The peptide was found to insert into bilayers at the interface level, without destroying the membrane, and showing a dominant aggregated �-sheet secondary structure with a marked tilt relative to the membrane surface, in a way very similar to cateslytin (vide supra). These results shine light in situations where overproduction of anti-apoptotic Bcl-2 plays a key role in the regulation of apoptotic path-ways in cancer. The fact that BH4 interacts with mitochon-drion external lipid membranes and converts them into ag-gregated structures, i.e., increases the order of the mem-branes and therefore their stiffness, could play a role in the regulation of apoptosis. Such a process could interfere with the anchoring of the pro-apoptotic proteins into the outer mitochondrial membrane system (vide supra, Bax-�1) and suggests that the anti-apoptotic specificity of the entire Bcl-2 protein could arise from strong interactions with the mito-chondrial membrane lipid: a process that would act as a ‘pro-tective’ mechanism against pore forming proapoptotic pro-teins.

Fig. (4). Model for interaction of cateslytin with bacterial-like membrane domains. Peptides are unstructured in solution (D) and adopt a �-sheet conformation upon interaction with negatively charged membranes (C). At the membrane, peptides aggregate to form �-sheet plates (antiparallel, right hand side insert). Passage across the membrane to alter biosynthetic pathways occurs through phase boundary defects (B)between thin (A) and thick (C) membrane domains (71). Bottom insert: side views from molecular dynamics calculations of the disordered pore formed at phase boundaries. The bilayer is represented by the position of the phosphate groups (green golden beads); fatty acyl chains are not shown. Water molecules (orange) in the vicinity of the pore are also shown (other water molecules are not shown). Peptides that par-ticipate to the pore are represented as ribbons (residues in green are polar, Arg) (71).

Outer cell

Inner cell

Biosyntheticpathways

A B C

D


Cysteine-Stabilized Alpha-Beta Peptides, CS��

Peptides that contain both the �-helical and �-strand structures are found in the world of defensins, i.e., peptides that are primarily generated by insects, but also by scorpions, molluscs and plants, as a first line of defence against bacte-rial, fungi or virus aggression [76-78]. Defensins are weakly cationic peptides, composed of 18–51 residues, including 6 to 8-conserved cysteine residues all of which participate in intramolecular disulfide bonds [5]. They lock the structure in a very particular globular fold as shown in Fig. (1), where only one �-helix is opposite to 2-3 �-strands. They are called cysteine-stabilized alpha-beta peptides, CS��. The alpha helix is not particularly amphipathic but the globular struc-ture is, offering one face hydrophobic, the other hydrophilic and weakly charged Fig. (1). Their activity against bacteria, fungi and some viruses is in the �M range but they are weakly cytotoxic. CS�� peptides apparently disrupt the permeability barrier of the cytoplasmic membrane of the gram-positive bacteria within seconds. On the basis of patch-clamp experiments on giant liposomes, defensin oligomers would form channels in the cytoplasmic membrane. How-ever, as for the barrel stave model proposed mechanism for �-helices, there is at present no atomic evidence of such a structure for CS��.

Cyclic Peptides

Some antimicrobials are now being considered as alterna-tive antibiotics, such as those made by bacteria and contain-ing small ring structures closed by a thioether bond. The attractive features of some of these peptides, i.e., their natu-ral sources, wide range of activities, ease of production, and the fact that they are not inducing the development of resis-tance, have established a wide interest in seeking for new antibiotics. Such a group of peptides is called lantibiotics and their structure and properties have recently been reviewed [79, 80]. One of the lantibiotics, Nisin is currently used as an antimicrobial agent for food preservation, possesses 34 resi-dues and belongs to group A lantibiotics with activity in the nM range against Gram-positive bacteria. It has been found that Nisin rapidly induces leakage of ions and small metabo-lites from bacteria and opens electrical conductance channels in black lipid membranes. Although it is proposed that Nisin forms a pore in a way similar to �-helical peptides, there it at present no structural evidence of such a mechanism, at the atomic level. However the special high affinity interaction of this peptide with a specific lipid, Lipid II, a precursor in the bacterial cell wall synthesis makes Nisin a special case among cyclic peptides. Extensive reviews on lantibiotics and Nisin can be found elsewhere [1, 80] and we will rather con-centrate on other cyclic peptides for which molecular mechanisms have been discovered. Another interesting set of cyclic peptides is the lipopep-tide family. The main difference between lipopeptides and more classical peptides is the presence of a hydrocarbon chain in complement to the peptidic small cyclic backbone. Most described peptides (mainly from Bacillus species), in-clude Polymyxin, Iturin, Fengycin, and Surfactin (SF). The latter is secreted by Bacillus subtilis and is composed of a ring-shaped peptidic backbone of seven amino acids (Glu LeuLeuValAspLeuLeu) in a chiral sequence of L/D enanti-

omers [81]. The ring is closed by a �-hydroxy fatty acid with different aliphatic chains of length ranging from 12 to 14 carbon atoms Fig. (1). The presence of both lipophilic and hydrophilic/negatively charged sections confers to SF pow-erful surfactant capabilities such as emulsification and foam-ing. It has a very large spectrum of biological activities in-cluding inhibition of fibrin clot formation, antibacterial, an-timycoplasmic, antitumoral, antiviral actions and is a hypo-cholesterolemicagent [82]. Surprisingly, this negatively char ged amphipathic peptide is very active against negatively charged membranes, at physiological pH, which may appear a little bit peculiar. However, the minute mechanism could be deciphered using solid state NMR, electron microscopy, molecular dynamics and dynamic light scattering to follow the action on SF on negatively charged model membranes mimicking Mycoplasma membranes against which SF is active [11]. Action of SF on negatively charged �m-size multilamellar liposomes (MLV) converts them into very small unilamellar vesicles, SUV, of 30-50 nm. As depicted in Fig. (5) this occurs in two steps, the first one is driven by the lipophilic favourable interaction be-tween the hydrocarbon SF tail and lipophilic amino acids with lipid chains. Then in a second step, an electrostatic re-pulsion between the negatively charged Asp and Glu resi-dues and the negative charges of lipids promotes consider-able curvature constraints leading to MLV “explosion” and stabilization of small vesicles. Such a mechanism is referred to as an “electrostatic wedge” because of the peculiar wedge shape of Surfactin when sitting in the membrane Fig. (5 in-sert) and is effective for very low doses on negatively charged membranes. Such a small vesicle formation does not occur when the membrane is zwitterionic as in mammals. It is worth mentioning other wedge models similar to that de-scribed above [83].

Short Linear Peptides

The above membrane interacting peptides are hydropho-bic enough to partition essentially into the membrane. Such a localization is primarily related to their length, greater than 16 amino acids, and to the elevated amount of lipophilic amino acids present in the sequence. There are however very small peptides that do affect membrane mechanisms such as neurotransmission and that partition almost equally into the membrane and in the water medium. In general these pep-tides contain fewer amino acids and bind to membrane re-ceptors to trigger or alter function. The purpose of this sec-tion is not to review short peptide binding to receptors but rather explore the current hypothesis that some short pep-tides must pass through the lipid core to adopt the active conformation leading to receptor adequate binding. One will take the example of enkephalins that are neurotransmitters found in the human central nervous system, especially in regions of the brain and spine associated with diffuse pain pathways. These opiate pentapeptides have the same recep-tors as morphine. In addition to the central control of respira-tion, they work to inhibit pain signals. Enkephalins are com-posed of five amino acids with the following sequence: Tyr-Gly-Gly-Phe-(Met or Leu) [25, 84]. It is believed that these neuropeptides interact with the nerve cell membrane in order to adopt a bioactive conformation that will then fit onto the receptors. The Fig. (6) reports the end result of a comprehen-


sive study involving structural determination of both the membrane and water 3D structure of Menk (Methionine Enkephalin) using solid and liquid state NMR and molecular dynamics [25]. In the case of the membrane embedded peptide Magic Angle Sample Spinning NMR was used to resolve individual amino acid resonances leading to complete structural deter-mination at atomic level. In solution, the peptide does not possess any structure whereas it shows a bent folding within the membrane. Not surprisingly the peptide sits at the mem-brane interface of a phosphatidylcholine (PC) lipid, with however the most hydrophobic amino acids pointing towards the membrane core. Such a finding is in agreement with the mechanism called ‘‘membrane catalysis’’ where the polar headgroups of the lipids at the cell membrane surface inter-act with these small peptides that enter into the membrane via hydrophobic interactions and undergo a conformational change. According to this mechanism enkephalins would migrate to the receptor site with the suitable structure for binding. In the light of further studies [85], it appears that the depth of insertion of Menk in the membrane systems is modulated by the composition of the bilayers such as PC, PE, PS, and PG and by pH conditions. More specifically, the insertion of enkephalins in membranes depends on the bal-ance between electrostatic and hydrophobic interactions. One may then conjecture that Menk would show a “structural plasticity”, i.e., adopt slightly different conformations ac-cording to the lipid composition of the membrane.

Models for Interaction: A Critical Viewpoint

From the above, it appears clearly that peptides may exert a wide variety of actions on membranes. Membrane peptides either adopt the functional structure within the membrane or modulate its stability though increase or decrease of mem-brane thickness. Such mechanisms are well documented in

the hydrophobic mismatch concept, early named as “mat-tress” model: the lipids of the peptides adapt their hydropho-bic length or tilt to minimize the exposure of hydrophobic parts to hydrophilic media. The lipid bilayer dynamics is modified, thickened or thinned, lateral domain formation occurs, but the membrane is never destroyed. The class of antibiotic peptides is very rich in structures (helices, �-strands, CS��, cyclic and/or lipopeptides) and functions (membrane permeation, membrane destruction, membrane-forming domains, etc.). They act very strongly on membranes and may lead to complete destruction or induce transient or permanent pores through which internal cell ma-terials will escape. Models for direct membrane lysis, i.e.,complete membrane restructuration to form new peptide lipid entities such as very small vesicles, discs or micelles, is based on hydrophobic interaction and/or electrostatic effects. The “carpet” mechanism where a sufficient concentration of marked amphipathic helices at the membrane surface segre-gate to cut the membrane into pieces or exert a considerable curvature tension through both hydrophobic plus electro-static repulsion (cyclic lipopeptides) appears to be backed up with abundant structural data, concerning at least the end product: nanometre discs, micelles and vesicles. The precise transitory mechanism, though very plausible, still lacks ex-perimental evidence. On the other hand the permeation mechanism, i.e., the formation of permanent or transient pores, is questionable. Although electrophysiology or leak-age measurements indicate clearly the opening of membrane “holes”, their precise structure may vary considerably. The very nice representation of the “barrel stave” model where amphipathic helices make a well-organized toroid structure is, to our knowledge, not backed up with structural experi-mental data at atomic resolution. The only available indica-tion where both electrophysiology and all atom molecular dynamics report the measurement of the same transmem-

Fig. (5). Artist view illustrating the ‘‘electrostatic wedge’’ model: SF molecules penetrate into the hydrophobic core of negatively charged microbial membranes because of favourable hydrophobic forces, and in a second step an electrostatic repulsion occurs between negative charges of peptidic amino acids Glu/Asp and negatively charged lipid headgroups. Such an effect promotes a strong increase in local mem-brane curvature, which destabilizes MLV (left hand side) to form SUVs (right hand side). Insert zoom shows details from molecular dynam-ics simulations: Surfactin, mainly in blue balls, inserts into the lipid membrane, stick representation, by means of its aliphatic tail and face.


brane current is for a �-stranded peptide that makes a disor-dered pore using a molecular electroporation mechanism. One must however stress that such structural data on peptide aggregates in the membrane is very hard to obtain because of the very unstable nature of such pores. They cannot be crys-talized like membrane proteins forming functional pores with helix bundles or beta barrels.

Fig. (6). Structures and locations of Methionine-enkephalin (Menk) peptide in water (linear) and at the bilayer interface (bent structure) as reconstructed from 2D-NMR constraints and molecular dynam-ics. Color code: peptide in ball representation; lipids in stick repre-sentation. Water molecules are not represented for clarity. From (25).

CONCLUSION

Membrane interacting peptides represent a very impor-tant class of molecules. Their moderate size, between 5 to 50 amino acids, and their amphipathic character provides them with considerable power in modulating biological membrane properties. The variability in lipid structure (alphabet of ca. 1000 letters obtained by combining heads and chains in glycero-, sphingo-, and sterols lipids) combined with that of peptide structure (20-40 letters by considering unnatural and modified amino acids) makes a dialog for both species that

involves considerable variability in structures and functions. Membrane peptides action is multiform and the structure and dynamics they adopt upon contact with membrane lipids varies considerably but depend in reality on a few physico-chemical concepts. The lipid membrane is an anisotropic amphipathic, charged and dynamic medium and the peptides are also amphipathic, charged and offer a wide structural plasticity. Electrostatic, van der Walls forces and hydrogen bonding are thus sufficient in driving interaction with mem-branes and account for the new lipid-peptide complexes formed. Due to this complexity, it is suggested that construc-tion of phase diagrams (temperature-composition) would help in defining all macromolecular events (new phases) that may happen upon peptide-membrane interaction [86].

CONFLICT OF INTEREST

The author(s) confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENT

Declared none.

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Received: February 11, 2011 Revised: May 09, 2011 Accepted: June 14, 2011

membrane interacting peptides: from killers to helpers

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