Amphipathic Helical Structure Research Articles

Quaternized 1,2,3-Triazolyl Content and Modulation Potentiate Antibacterial and Antifungal Activities of Amphipathic Peptoids.

Bioinspired from cationic antimicrobial peptides, sequence-defined triazolium-grafted peptoid oligomers (6- to 12-mer) were designed to adopt an amphipathic helical polyproline I-type structure. Their evaluation on a panel of bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis), pathogenic fungi (Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus), and human cells (hRBC, BEAS-2B, Caco-2, HaCaT, and HepG2) enabled the identification of two heptamers with improved activity to selectively fight Staphylococcus aureus pathogens. Modulation of parameters such as the nature of the triazolium and hydrophobic/lipophilic side chains, the charge content, and the sequence length drastically potentiates activity and selectivity. Besides, the ability to block the proinflammatory effect induced by lipopolysaccharide or lipoteichoic acid was also explored. Finally, biophysical studies by circular dichroism and fluorescence spectroscopies strongly supported that the bactericidal effect of these triazolium-grafted oligomers was primarily due to the selective disruption of the bacterial membrane.

Retention time prediction for post-translationally modified peptides: Ser, Thr, Tyr-phosphorylation

The development of a peptide retention prediction model for reversed-phase chromatography applications in proteomics is reported for peptides carrying phosphorylated Ser, Thr and Tyr-residues. The major retention features have been assessed using a collection of over 10,000 phosphorylated/non-phosphorylated peptide pairs identified in a series 1D and 2D LC-MS/MS acquisitions using formic acid as ion pairing modifier. Single modification event on average results in increased peptide retention for phosphorylation of Ser (+ 1.46), Thr (+1.33), Tyr (+0.93% acetonitrile, ACN) on gradient elution scale for Luna C18(2) stationary phase. We established several composition and sequence specific features, which drive deviations from these average values. Thus, single phosphorylation of serine results in retention shifts ranging from -2.4 to 5.5% ACN depending on position of the residue, nature of nearest neighbour residues, peptide length, hydrophobicity and pI value, and its propensity to form amphipathic helical structures. We established that the altered ion-pairing environment upon phosphorylation is detrimental for this variability. Hydrophobicity of ion-pairing modifier directly informs the magnitude of expected shifts: (most hydrophilic) 0.5 % acetic acid (larger positive shift upon phosphorylation) > 0.1 % formic acid (positive) > 0.1 % trifluoroacetic (negative) > 0.1 % heptafluorobutyric acid (larger negative shift). The effect of phosphorylation has been also evaluated for several separation conditions used in the first dimension of 2D LC applications: high pH reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), strong cation- and strong anion exchange separations.

Introduction of constrained Trp analogs in RW9 modulates structure and partition in membrane models

Over the past decades, many cell-penetrating peptides (CPP) have been studied for their capacity to cross cellular membranes, mostly in order to improve cellular uptake of therapeutic agents. Even though hydrophobic and anionic CPPs have been described, many of them are polycationic, due to the presence of several arginine (Arg) residues. Noteworthy, however, the presence of aromatic amino acids such as tryptophan (Trp) within CPPs seems to play an important role to reach high membranotropic activity. RW9 (RRWWRRWRR) is a designed CPP derived from the polyarginine R9 presenting both features. In general, when interacting with membranes, CPPs adopt an optimal conformation for membrane interactions – an amphipathic helical secondary structure in the case of RW9. Herein, we assumed that the incorporation of a locally constrained amino acid in the peptide sequence could improve the membranotropic activity of RW9, by facilitating its structuration upon contact with a membrane, while leaving a certain plasticity. Therefore, two cyclized Trp derivatives (Tcc and Aia) were synthesized to be incorporated in RW9 as surrogates of Trp residues. Thus, a series of peptides containing these building blocks has been synthesized by varying the type, position, and number of modifications. The membranotropic activity of the RW9 analogs was studied by spectrofluorescence titration of the peptides in presence of liposomes (DMPG), allowing to calculate partition coefficients (Kp). Our results indicate that the partitioning of the modified peptides depends on the type, the number and the position of the modification, with the best sequence being [Aia4]RW9. Interestingly, both NMR analysis and molecular dynamic (MD) simulations indicate that this analog presents an extended conformation similar to the native RW9, but with a much-reduced structural flexibility. Finally, cell internalization properties were also confirmed by confocal microscopy.

Role of N17's hydrophobic face in membrane curvature sensing and polyQ aggregation.

N17, the N-terminal domain of Huntingtin protein, assumes an amphipathic helical structure when in contact with membranes and in aggregate form, having one hydrophobic face and one hydrophilic face. Studies on N17 have underscored its importance as a membrane binding domain and its significance in the aggregation process.

Chromatographic behaviour of peptides modified with amine-reacting tags for relative protein quantitation in proteomic applications

Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts ΔHI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs.

Effects of charge on the interaction of the huntingtin N-terminal sequence with model cell membranes

Huntington’s Disease (HD) is a genetic neurodegenerative disorder caused by an expansion in the polyglutamine (poly-Q) region near the N-terminus of the huntingtin (htt) protein. When the poly-Q expansion surpasses a threshold of 35-40 glutamine repeats, the protein misfolds into a beta sheet rich structure that forms insoluble neuronal aggregates. The poly-Q region of htt is flanked by a 17 amino acid sequence (Nt17) that facilitates insertion of htt into lipid membranes via an amphipathic alpha helical structure, a function that is proposed to anchor the protein and may affect the aggregation process.

Peptide retention time prediction for peptides with post-translational modifications: N-terminal (α-amine) and lysine (ε-amine) acetylation

Development of a peptide retention prediction model in reversed-phase chromatography is reported for acetylated peptides – both N-terminal (α-) and side chain of Lys (ε-amine) residues. Large-scale proteomic 2D LC-MS analyses of acetylated/non-acetylated tryptic digest of whole human cell lysate have been used to assemble representative retention data sets of 25,000+ modified/non-modified pairs. This allowed elucidating chromatographic behaviour of modified peptides in three different separation modes: high pH reversed-phase, HILIC separation on amide phase (first dimension of 2D) and reversed-phase separation with formic acid as ion-pairing modifier in the second dimension. On average, N-terminal acetylation increases peptide RP retention at acidic pH by 5 Hydrophobicity Index units (% acetonitrile). Acetylation of first lysine adds another 4.1%. The magnitude of the retention shift varies greatly depending on the number of modified amines, peptide length, and N-terminal peptide sequence. Large retention shifts have been observed for peptides with hydrophobic N-termini and specifically peptides carrying sequences characteristic for amphipathic helical structures – all in complete agreement with major sequence-specific features of RP retention mechanism. The utility of the modified Sequence Specific Retention Calculator model has been verified for the in-vivo N-terminally acetylated peptides detected by 2D LC-MS/MS analysis of a yeast tryptic digest. The effect of N-terminal acetylation was also evaluated for six different HILIC columns, strong cation- and strong anion exchange separations using previously acquired 2D LC-MS/MS data.

Apolipoprotein Mimetic Peptides: An Emerging Therapy against Diabetic Inflammation and Dyslipidemia.

Obesity has achieved epidemic status in the United States, resulting in an increase in type 2 diabetes mellitus, dyslipidemia, and cardiovascular disease. Numerous studies have shown that inflammation plays a key role in the development of insulin resistance and diabetic complications. HDL cholesterol levels are inversely associated with coronary heart disease in humans. The beneficial effect of HDL is due, in part, to apolipoproteins A-I and E, which possess anti-inflammatory properties. The functional quality of HDL, however, may be reduced in the context of diabetes. Thus, raising levels of functional HDL is an important target for reducing inflammation and diabetic complications. Apo A-I possesses eight alpha-helical sequences, most of which form class A amphipathic helical structures. Peptides belonging to this class inhibit atherogenesis in several mouse models. Additional peptides based on structural components of apoE have been shown to mediate a rapid clearance of atherogenic lipoproteins in dyslipidemic mice. In this review, we discuss the efficacy of apolipoprotein mimetic peptides in improving lipoprotein function, reducing inflammation, and reversing insulin resistance and cardiometabolic disease processes in diabetic animals.

Membrane binding properties of the C-terminal segment of retinol dehydrogenase 8

Light absorption by rhodopsin leads to the release of all-trans retinal (ATRal) in the lipid phase of photoreceptor disc membranes. Retinol dehydrogenase 8 (RDH8) then reduces ATRal into all-trans retinol, which is the first step of the visual cycle. The membrane binding of RDH8 has been postulated to be mediated by one or more palmitoylated cysteines located in its C-terminus. Different peptide variants of the C-terminus of RDH8 were thus used to obtain information on the mechanism of membrane binding of this enzyme. Steady-state and time-resolved fluorescence measurements were performed using short and long C-terminal segments of bovine RDH8, comprising one or two tryptophan residues. The data demonstrate that the amphipathic alpha helical structure of the first portion of the C-terminus of RDH8 strongly contributes to its membrane binding, which is also favored by palmitoylation of at least one of the cysteines located in the last portion of the C-terminus.

Computational Study of Helical and Helix-Hinge-Helix Conformations of an Anti-Microbial Peptide in Solution by Molecular Dynamics and Vibrational Analysis.

Many classical antimicrobial peptides adopt an amphipathic helical structure at a water-membrane interface. Prior studies led to the hypothesis that a hinge near the middle of a helical peptide plays an important role in facilitating peptide-membrane interactions. Here, dynamics and vibrations of a designed hybrid antimicrobial peptide LM7-2 in solution were simulated to investigate its hinge formation. Molecular dynamics simulation results on the basis of the CHARMM36 force field showed that the α-helix LM7-2 bent around two or three residues near the middle of the peptide, stayed in a helix-hinge-helix conformation for a short period of time, and then returned to a helical conformation. High-resolution computational vibrational techniques were applied on the LM7-2 system when it has α-helical and helix-hinge-helix conformations to understand how this structural change affects its inherent vibrations. These studies concentrated on the calculation of frequencies that correspond to backbone amide bands I, II, and III: vibrational modes that are sensitive to changes in the secondary structure of peptides and proteins. To that end, Fourier transforms were applied to thermal fluctuations in C-N-H angles, C-N bond lengths, and C═O bond lengths of each amide group. In addition, instantaneous all-atom normal mode analysis was applied to monitor and detect the characteristic amide bands of each amide group within LM7-2 during the MD simulation. Computational vibrational results indicate that shapes and frequencies of amide bands II and especially III were altered only for amide groups near the hinge. These methods provide high-resolution vibrational information that can complement spectroscopic vibrational studies. They assist in interpreting spectra of similar systems and suggest a marker for the presence of the helix-hinge-helix motif. Moreover, radial distribution functions indicated an increase in the probability of hydrogen bonding between water and a hydrogen atom connected to nitrogen (HN) in such a hinge. The probability of intramolecular hydrogen bond formation between HN and an amide group oxygen atom within LM7-2 was lower around the hinge. No correlation has been found between the presence of a hinge and hydrogen bonds between amide group oxygen atoms and the hydrogen atoms of water molecules. This result suggests a mechanism for hinge formation wherein hydrogen bonds to oxygen atoms of water replace intramolecular hydrogen bonds as the peptide backbone folds.

The structure and antimicrobial potential of wasp and hornet (Vespidae) mastoparans: A review

AbstractWasp venom is a complex mixture of biologically active components, including high molecular weight proteins, small peptides, bioactive amines, and amino acids. Peptides comprise up to 70% of dried venom. In social wasp venoms, three of the major peptide types are mastoparans, which cause mast cell degranulation, chemotactic peptides, which promote chemotaxis of polymorphonucleated leukocytes, and kinin‐related peptides, which are known to produce pain and increase vascular permeability. Among these, the bioactive tridecapeptide mastoparan is the most common and may even have antimicrobial activity. Herein we summarize the results of studies on vespid mastoparans, focusing on hornets (Vespa spp.) identified following a systematic literature search for mastoparans of hornets in the genus Vespa, the most active mastoparan research taxon. The common features of hornet mastoparans are C‐terminal amidation, amphipathic helical structure, and multiple functions such as mast cell degranulation and hemolysis, as well as membrane permeabilization. Most interestingly, all tested hornet mastoparans have strong antimicrobial activities, suggesting that they can provide useful insights into and opportunities for development of novel antibacterial peptides.

Effect of targeted minimal sequence variations on the structure and biological activities of the human cathelicidin LL‐37

AbstractLL‐37 is an innate immune peptide derived from the human cathelicidin, which exerts pleiotropic roles in host defense and healing. These activities in part depend on its capacity to adopt an amphipathic helical structure in physiological solutions and then oligomerizing. Orthologues from other primates, such as rhesus RL‐37, remain monomeric and disordered under the same conditions. Intramolecular salt‐bridges, arising from appropriately spaced anionic and cationic residues in its sequence, may play a relevant role in determining the particular structure adopted by LL‐37. To probe this, we have effected minimal, targeted residue variations such as replacement of a single residue (K15→G), or inversion of one or both sets of two residues (E10 K11→ K10 E11 or E16 K18→ K16 E18). This could alter the pattern of intramolecular salt bridging without affecting other functionally relevant parameters such as overall hydrophobicity, helix amphipathicity or charge. The structural and functional effects were analyzed using CD spectroscopy, surface plasmon resonance, antimicrobial activity assays, and bacterial membrane permeabilization to fluorescent probes of increasing sizes, using flow cytometry. Analogs were functionally different from both LL‐37 and RL‐37, so it was not possible to switch from the function of one to that of the other simply by altering the salt‐bridging pattern in this manner. This indicates that the particular structure/function characteristics of LL‐37 likely depend quite subtly, and in a precise and complex manner, on a complex pattern of intramolecular interactions.

Marsupial and monotreme cathelicidins display antimicrobial activity, including against methicillin-resistant Staphylococcus aureus.

With the growing demand for new antibiotics to combat increasing multi-drug resistance, a family of antimicrobial peptides known as cathelicidins has emerged as potential candidates. Expansions in cathelicidin-encoding genes in marsupials and monotremes are of specific interest as the peptides they encode have evolved to protect immunologically naive young in the harsh conditions of the pouch and burrow. Our previous work demonstrated that some marsupial and monotreme cathelicidins have broad-spectrum antibacterial activity and kill resistant bacteria, but the activity of many cathelicidins is unknown. To investigate associations between peptide antimicrobial activity and physiochemical properties, we tested 15 cathelicidin mature peptides from tammar wallaby, grey short-tailed opossum, platypus and echidna for antimicrobial activity against a range of bacterial and fungal clinical isolates. One opossum cathelicidin ModoCath4, tammar wallaby MaeuCath7 and echidna Taac-CATH1 had broad-spectrum antibacterial activity and killed methicillin-resistant Staphylococcus aureus. However, antimicrobial activity was reduced in the presence of serum or whole blood, and non-specific toxicity was observed at high concentrations. The active peptides were highly charged, potentially increasing binding to microbial surfaces, and contained amphipathic helical structures, which may facilitate membrane permeabilisation. Peptide sequence homology, net charge, amphipathicity and alpha helical content did not correlate with antimicrobial activity. However active peptides contained a significantly higher percentage of cationic residues than inactive ones, which may be used to predict active peptides in future work. Along with previous studies, our results indicate that marsupial and monotreme cathelicidins show potential for development as novel therapeutics to combat increasing antimicrobial resistance.

Apolipoprotein Mimetic Peptides as Modulators of Lipoprotein Function.

Apolipoprotein (apo)A-I and apoE are the two protein components that have been extensively investigated for their anti-atherogenic properties. Both apolipoproteins possess amphipathic helical structures, responsible for the solubilization of lipids. While apoA-I possesses class A amphipathic helical structures, apoE possesses a 59 residue long amphipathic helical domain linked to a four helix bundle containing the Arg-rich, 10 residue receptor binding domain. An 18 residue model peptide (18A) was designed to mimic the amphipathic helical domains of apoA-I. This and several analogs were able solubilize phospholipids and, when administered into animal models of atherosclerosis, were able to inhibit lesion formation without any effect on plasma cholesterol levels. These analogs were subsequently termed as apoA-I mimetic peptides. When this peptide (18A) was covalently linked to the Arg-rich receptor binding domain of apoE, the resulting peptide Ac-hE18A-NH2, in which hE refers to the 141-150 Arg-rich region of apoE, dramatically reduced plasma cholesterol in several dyslipidemic animal models, resulting in the reduction of lesion formation. This and several other analogs which were able to dramatically decrease plasma cholesterol, analogous to apoE, were termed as apoE mimetic peptides. These observations developed the field of apolipoprotein mimetic peptides which are involved in interacting with lipoproteins and modulating their function. The present review describes progress made in this field which have culminated in clinical trials in humans for both the apoA-I and apoE mimetic peptides.

Abstract 328: Antitumor activity of the oncolytic peptide LTX-315 in syngeneic tumor models

Abstract Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in a number of species as a defense mechanism for eukaryotic cells against pathogens and are an integral part of the innate immune system. CAPs have a broad spectrum of activities including antimicrobial and anticancer while being less cytotoxic toward non-malignant cells. The potential therapeutic application against cancer has spawned an interest in developing oncolytic agents that display a new mode of action to overcome the potential drug resistance associated with other current therapeutics. The anticancer effects of CAPs are still under investigation, but several peptides have already exhibited a promising potential with cytotoxic activities against a broad spectrum of tumor cells. Oncolytic peptides exert their activity through either a membranolytic mode of action or an interaction with intracellular targets, or a combination of both. LTX-315 (K-K-W-W-K-K-W-Dip-K-NH2), a novel oncolytic peptide developed by Lytix Biopharma AS has the potential to adopt an amphipathic helical coil structure. In vitro studies have demonstrated that it was highly effective against both drug-resistant and drug-sensitive cancer cells from several organ origins, with lower toxicity toward normal cells. LTX-315 was designed for intratumoral treatment of transdermal lesions. Previously, LTX-315 has been shown to induce complete regression of B16 melanomas and long lasting antitumor immune responses. Histological analyses of treated tumors revealed extensive hemorrhagic necrosis and infiltration of CD3+ T cells. Moreover, mRNA levels of inflammatory cytokines such as IL1β, IL6 and IL18 were found to be increased in the tumor tissue after LTX-315 treatment. The treatment did also prevent lung metastasis in mice re-challenged with B16F1 cells intravenously. Due to its oncolytic mode of action, LTX-315 induces immunogenic cell death through the release of danger-associated molecular pattern molecules and tumor antigens. Recently, we have demonstrated that when subcutaneously established EMT-6 tumors (inoculated into both flanks of the animal) were treated intratumorally with LTX-315, an antitumor response was observed with a T/C ratio of 17% 19 days post start of treatment. Furthermore, an abscopal effect of LTX-315 on the untreated tumor was also reported but only when it was combined with anti-PD-L1 antibody. At the end of study, 50% of mice that had received the combination therapy were still alive vs 30% and 40% in the groups treated with LTX-315 or anti-PD-L1 antibody alone, respectively. In conclusion, LTX-315 seems to be an ideal combinations partner for immune checkpoint inhibitors. Citation Format: Ketil André Camilio, Baldur Sveinbjørnsson, Sylvie Maubant, Guillaume Serin, Jean-François Mirjolet, Francis Bichat, Øystein Rekdal. Antitumor activity of the oncolytic peptide LTX-315 in syngeneic tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 328.

Rational Evolution of Antimicrobial Peptides Containing Unnatural Amino Acids to Combat Burn Wound Infections.

Antimicrobial peptides have long been raised as a promising strategy to combat bacterial infection in burn wounds. Here, we attempted to rationally design small antimicrobial peptides containing unnatural amino acids by integrating in silico analysis and in vitro assay. Predictive quantitative sequence-activity models were established and validated rigorously based on a large panel of nonamer antimicrobial peptides with known antibacterial activity. The best quantitative sequence-activity model predictor was employed to guide genetic evolution of a peptide population. In the evolution procedure, a number of unnatural amino acids with desired physicochemical properties were introduced, resulting in a genetic evolution-improved population, from which seven peptide candidates with top scores, containing 1-3 unnatural amino acids, and having diverse structures were successfully identified, and their antibacterial potencies against two antibiotic-resistant bacterial strains isolated from infected burn wounds were measured using in vitro susceptibility test. Consequently, four (WL-Orn-LARKIV-NH2 , ARKRWF-Dab-FL-NH2 , KFI-Hag-IWR-Orn-R-NH2 and YW-Hag-R-Cit-RF-Orn-N-NH2 ) of the seven tested peptides were found to be more potent than reference Bac2A, the smallest naturally occurring broad spectrum antimicrobial peptide. Molecular dynamics simulations revealed that the designed peptides can fold into amphipathic helical structure that allows them to interact directly with microbial membranes.

Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages.

Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation.

Membrane Insertion Depth and Curvature Sensing

Many cellular processes require specific membrane deformations and reorganization, including endocytosis and cell division. Recently it has been shown that the degree of membrane curvature regulates activity and localization for some proteins such as ArfGAP1 and SpoVM. The short peptide SpoVM is one of the first proteins to localize to the forespore during Bacillus subtilis sporulation by recognizing the slightly curved convex membranes. However, it is unclear how a 26-amino acid protein can recognize curvature, but the unique topology of SpoVM and the phospholipid bilayer provide some insight into this process.The non-traditional amphipathic helical structure of SpoVM consists of a short helical region and a flexible N-terminal loop. The helix has a comparatively large non-polar surface, with a small polar surface. These features allow for insertion of the helix deep within the phospholipid bilayer while the relatively polar tail can extend out of the membrane. We will present in vivo and in vitro data of SpoVM variants in support of our hypothesis that SpoVM's ability to sense membrane curvature is critically dependent on successful deep membrane insertion.

Folded structure and insertion depth of the frog-skin antimicrobial Peptide esculentin-1b(1-18) in the presence of differently charged membrane-mimicking micelles.

Antimicrobial peptides (AMPs) are effectors of the innate immunity of most organisms. Their role in the defense against pathogen attack and their high selectivity for bacterial cells make them attractive for the development of a new class of antimicrobial drugs. The N-terminal fragment of the frog-skin peptide esculentin-1b (Esc(1-18)) has shown broad-spectrum antimicrobial activity. Similarly to most cationic AMPs, it is supposed to act by binding to and damaging the negatively charged plasma membrane of bacteria. Differently from many other AMPs, Esc(1-18) activity is preserved in biological fluids such as serum. In this work, a structural investigation was performed through NMR spectroscopy. The 3D structure was obtained in the presence of either zwitterionic or negatively charged micelles as membrane models for eukaryotic and prokaryotic membranes, respectively. Esc(1-18) showed a higher affinity for and deeper insertion into the latter and adopted an amphipathic helical structure characterized by a kink at the residue G8. These findings were confirmed by measuring penetration into lipid monolayers. The presence of negatively charged lipids in the bilayer appears to be necessary for Esc(1-18) to bind, to fold in the right three-dimensional structure, and, ultimately, to exert its biological role as an AMP.

Native oligomerization determines the mode of action and biological activities of human cathelicidin LL-37

LL-37 is a multifunctional component of innate immunity, with a membrane-directed antimicrobial activity and receptor-mediated pleiotropic effects on host cells. Sequence variations in its primate orthologues suggest that two types of functional features have evolved; human LL-37-like peptides form amphipathic helical structures and self-assemble under physiological conditions, whereas rhesus RL-37-like peptides only adopt this structure in the presence of bacterial membranes. The first type of peptide has a lower and more medium-sensitive antimicrobial activity than the second type, but an increased capacity to stimulate host cells. Oligomerization strongly affects the mode of interaction with biological membranes and, consequently, both cytotoxicity and receptor-mediated activities. In the present study we explored the effects of LL-37 self-association by using obligate disulfide-linked dimers with either parallel or antiparallel orientations. These had an increased propensity to form stacked helices in bulk solution and when in contact with either anionic or neutral model membranes. The antimicrobial activity against Gram-positive or Gram-negative bacteria, as well as the cytotoxic effects on host cells, strongly depended on the type of dimerization. To investigate the extent of native oligomerization we replaced Phe5 with the photoactive residue Bpa (p-benzoyl-L-phenylalanine), which, upon UV irradiation, enabled covalent cross-linking and allowed us to assess the extent of oligomerization in both physiological solution and in model membranes.