Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion

UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC); and their secondary structure was determined by circular dichroism (CD). Three different lipid systems were used to mimic red blood cells, Escherichia coli and Staphylococcus aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not hemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against Acinetobacter baumanni, and candidates for peptidomimetics to enhance their potency and minimize hemolysis. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Solution structures and biological functions of the antimicrobial peptide, arenicin-1, and its linear derivative

Arenicin-1 (AR-1) is a novel antimicrobial peptide that was isolated from coelomocytes of the marine polychaeta lugworm Arenicola marina and shown to contain a single disulfide bond between Cys3 and Cys20, forming an 18-residue ring [Ovchinnikova, T. V. et al., FEBS Lett 2004, 577, 209-214]. To determine the role of this disulfide bond, we synthesized AR-1 (RWCVYAYVRVRGVLVRYRRCW) and its linear derivative, arenicin-1-S (AR-1-S: RWSVYAYVRVRGVLVRYRRSW). Activity assays revealed that AR-1-S is somewhat less active against bacterial cells than AR-1. Both peptides were very hydrophobic, and displayed cytotoxicity against human red blood cells. Analysis of the tertiary structures of AR-1 and AR-1-S by NMR spectroscopy disclosed that AR-1 has two-stranded antiparallel beta-sheet structures with amphipathicity, while AR-1-S displays a random coil structure in DMSO. Our biological data for AR-1 and AR-1-S indicate that the hydrophobic-hydrophilic balance, disulfide bridge, and the amphipathic beta-sheet structure of the peptides play important roles in their biological activities. Elucidation of the structure of AR-1 and its derivative should facilitate the design of novel non-cytotoxic peptide antibiotics with potent antibacterial activities.     

Comparison of NMR structures and model-membrane interactions of 15-residue antimicrobial peptides derived from bovine lactoferricin.

LFB (FKCRRWQWRMKKLGA-HN2) is a 15-residue linear antimicrobial peptide derived from bovine lactoferricin, which has antimicrobial activity similar to that of the intact 25-residue disulfide-cyclized peptide. Previous alanine-scan studies, in which all of the residues in LFB were individually replaced with Ala, showed that the 2 tryptophan (Trp) residues of LFB were crucial to its antimicrobial activity. When either Trp6 or Trp8 was replaced with Ala (LFBA6 and LFBA8, respectively), these 2 peptides were almost devoid of antimicrobial activity. We determined the structures of LFB, LFBA6, and LFBA8 bound to membrane-mimetic SDS micelles using NMR spectroscopy, and studied their interactions with different phospholipid-model membranes. The membrane interactions of LFB exhibited little correlation with its antimicrobial activity, suggesting that the mechanism of action of LFB involves intracellular targets. However, the much higher antimicrobial activity of LFB compared with LFBA6 and LFBA8 might result, in part, from the formation of energetically favorable cation-pi interactions observed only in LFB. Information about the importance of Arg and Trp cation-pi interactions will provide insight for the future design of potent antimicrobial peptidomimetics.     

NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.     

Cyclic analogs of insect oostatic peptides: synthesis, biological activity, and NMR study

Cyclic peptides 2a-2c, derived from the sequence of the C-terminal shortened analogs of the oostatic decapeptide H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a), were synthesized and assayed on their effect in a reproduction of the flesh fly Neobellieria bullata. The cyclization of the N-terminal linear tetra- and pentapeptides 1b and 1c to the cyclotetra- and cyclopentapeptides 2b and 2c decreased the oostatic activity by one order of magnitude. The cyclodecapeptide 2a, which emerged spontaneously during the pentapeptide cyclization, was quite inactive. Comparative 1H and 13C NMR study on a conformation of the cyclopeptides 2a-2c, and their linear precursors 1b and 1c revealed that a space structure of the cyclic analogues 2b and 2c is too restricted to adopt a biological conformation necessary for receptor binding and therefore only minor oostatic activity is observed after their application. The lack of the oostatic activity in the case of the more flexible dimeric analogue 2a is ascribed to the size of its molecule and its overall shape that is not compatible with a receptor binding.     

CD and NMR structural characterization of ceratotoxins,natural peptides with antimicrobial activity

Antibacterial properties of the secretion from the female reproductive accessory glands of medfly Ceratitis capitata are mostly ascribed to the presence of two peptides, ceratotoxin A and B, which exhibit a strong activity against gram-positive and gram-negative bacterial strains, and show sequence and function homology with cecropins, melittin, and magainins. CD experiments performed in different solvents indicate the presence of a significant content of helical structures in organic solvent. Two-dimensional nmr results for ceratotoxin A in methanol show a helical behavior for the 8–25 region of the peptide. A Ramachandran classification of each residue for the structures obtained from distance geometry calculations lead to the definition of four structural families in which the central segment 10–19 is always helical and differences refer to residues 8–9 and 19–23. A sequence analysis of the two ceratotoxins and a systematic search on the protein data bank revealed the occurrence of a KX-hydrophobic-hydrophobic-P motif that seems to be important for helix stabilization. © 1996 John Wiley & Sons, Inc.     

NMR evidence of hydrogen bonding in 1-ethyl-3-methylimidazolium-tetrafluoroborate room temperature ionic liquid

The 1-ethyl-3-methylimidazolium-tetrafluoroborate (EMI–BF₄) room temperature ionic liquid was investigated with NMR techniques. Diffusion coefficients measured at temperatures ranging from 300 to 360 K indicate that phase-change occurred in the vicinity of 333 K, which is supported by ¹¹B quadrupolar relaxation rates. This phase change is ascribed to the transformation of the diffusion particle from ‘discrete ion-pair’ to ‘individual ion’ at temperatures above 335 K due to decomposition of the EMI–BF₄ ion pair. Analysis of the ¹³C dipole–dipole relaxation rates identifies the formation of hydrogen bond (C2HF) between the counterions, EMI⁺ and BF₄ ⁻. This hydrogen bonding may have significant contribution to the higher viscosity of this ionic liquid in comparison with the EMI–AlCl₄ ionic liquid at corresponding temperatures.     

Influence of hydrogen bonding on the rotamer distribution of the histidine side chain in peptides: 1H NMR and CD studies.

Both 1H NMR and circular dichroism pH titration studies on histidine, His-Gly, Gly-His and Gly-His-Gly indicate that the side-chain spatial orientation depends strongly on the vicinal charges. The arrangement of the imidazole side-chain (rotamer population) is shown by the histidine beta and beta' and the glycine methylene proton chemical shifts as well as the vicinal 1H-1H coupling constants 3JCalpha-H-beta-H, beta'-H. For His-Gly and Gly-His-Gly a good correlation can be found between the ionization of the glycine COOH group and the increase of rotamer III (g-g) which is also visualized by circular dichroism through an enhancement of the ellipticity at 212 nm. In these two peptides a hydrogen bond between the imidazolium and the carboxylate group is supposed to stabilize rotamer III at pH 4-5.     

Solution structure and model membrane interactions of temporins-SH, antimicrobial peptides from amphibian skin. A NMR spectroscopy and differential scanning calorimetry study

Temporin-SHa and temporin-SHc are 13 residue long antimicrobial peptides from frog skin that have similar sequences but differ markedly in their membrane-damaging properties. Temporin-SHa contains a single basic lysine residue and has a unique antimicrobial spectrum of action among temporins, being very potent against Gram-positive and Gram-negative bacteria, yeasts, fungi, and protozoa. Temporin-SHc, which contains a single basic histidine residue, is inactive against Gram-negative bacteria, has a reduced efficacy against Gram-positive bacteria, but is still active against yeasts and fungi. Temporin-SHb, with no basic residue, has no antimicrobial activity. The three-dimensional structures of the peptides bound to SDS micelles were analyzed by CD and NMR spectroscopy combined with restrained molecular dynamics calculations. The peptides adopt well-defined amphipathic alpha-helical structures extending from residue 3 to residue 12, when bound to SDS micelles. The structures are stabilized by extensive interactions between aliphatic and aromatic side chains on the nonpolar face. Relaxation enhancements caused by paramagnetic probes showed that the peptides adopt nearly parallel orientations to the micelle surface and do not deeply penetrate into the micelle. The interaction of the peptides with model membranes was investigated by differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles and membrane-permeabilization assays on calcein-loaded large unilamellar vesicles. Calorimetric data indicated that both temporin-SHa and -SHc reside at the hydrocarbon core-water interface of the anionic lipid bilayer but interact with anionic bilayers in a very different manner. This suggests that the charge-induced activity of temporins-SH for bacterial cells is due to changes in the membrane-disturbing mechanism of the bound peptides.     

Solution structures of stomoxyn and spinigerin, two insect antimicrobial peptides with an alpha-helical conformation

Stomoxyn and spinigerin belong to the class of linear cysteine-free insect antimicrobial peptides that kill a range of microorganisms, parasites, and some viruses but without any lytic activity against mammalian erythrocytes. Stomoxyn is localized in the gut epithelium of the nonvector stable fly that is sympatric with the trypanosome vector tsetse fly. Spinigerin is stored and secreted by hemocytes from the fungus-growing termite. The structure of synthetic stomoxyn and spinigerin in aqueous solution and in TFE/water mixtures was analyzed by CD and NMR spectroscopy combined with molecular modeling calculations. Stomoxyn and spinigerin adopt a flexible random coil structure in water while both assume a stable helical structure in the presence of TFE. In 50% TFE, the structure of stomoxyn is typical of cecropins, including an amphipathic helix at the N-terminus and a hydrophobic C-terminus with helical features that probably fold in a helical conformation at higher TFE concentration. In contrast to stomoxyn, spinigerin acquires very rapidly a helical conformation. In 10% TFE the helix is highly bent and the structure is poorly defined. In 50% TFE, the helical structure is well defined all along its sequence, and the slightly bent alpha-helix displays an amphiphilic character, as observed for magainin 2. The structural similarities between stomoxyn and cecropin A from Hyalophora cecropia and between spinigerin and magainin 2 suggest a similar mode of action on the bacterial membranes of both pairs of peptides. Our results also confirm that TFE induces helix formation and propagation for amino acids showing helical propensity in water but also enhances the helix propagation propensity of nonpolar beta-branched residues.     

Three new supramolecular networks formed via hydrogen bonding interactions: Syntheses, crystal structures and magnetic properties

Three new iron(III) citrate complexes [Fe₂(cit)₂(H₂O)₂](H₂bpa) (1), [Fe₂(cit)₂(H₂O)₂](H₂bpe) (2) and [Fe₄(cit)₄(H₂O)₄](H₂bpp)₂(H₂O) (3) (cit = C(O⁻)(COO⁻)(CH₂COO⁻)₂, bpa = 1,3-bis(4-pyridyl)ethane, bpe = 1,3-bis(4-pyridyl)ethene, bpp = 1,3-bis(4-pyridyl)propane) were synthesized and characterized by elemental analysis, spectroscopic techniques and magnetic properties. Single X-ray diffraction analyses in the 1-3 complexes reveal that the iron ion is six-coordinated and is bound by two deprotonated citrates and a pair of aqua ligands in a distorted octahedral fashion. The anionic complex contains a centro-symmetrical planar of four-membered Fe₂O₂ ring. There are significant contributions to the stabilities of the assembled lattices in 1-3 arising from the protonated pyridine analogue counterions neutralizing the anionic charges of the complexes. The units in the complexes are connected together via hydrogen bonding to form 3D supramolecular networks. The supramolecular structures of 1-2 show alternating and motif linking the anionic moieties which are in turn interwoven with cationic moieties, while 3 shows alternating and motif. The magnetic properties of 1-3 are investigated and discussed in detail.     

In-silico analysis of Sirt2 from Schistosoma mansoni: structures,conformations, and interactions with inhibitors

Sirtuins are NAD+-dependent lysine deacetylases member of the class III HDAC family. These are demonstrated to be therapeutic targets in parasitic diseases like schistosomiasis. Observations suggested that sirtuin enzyme is necessary for the functionality of fe/male reproductive system, due to which SmSirt2 is treated as a potential therapeutic target. There are no structural and molecular features of SmSirt2 have been reported yet. In this study, homology modeling has been used to determine the three-dimensional features of the SmSITRT2. Further, structure validation has been performed by energy minimization and Ramachandran plot. Validated structures are further subjected to molecular docking and virtual screening to find the best lead molecules for downstream analysis. Ten lead molecules were selected while comparing virtual screening of hSirt2 and SmSirt2 both. These leads are further compared with AKG2 which is known inhibitor of hSirt2 (?8.8 kcal/mol). Out of selected 10 leads, four of them (ZINC23995485 (?9.5 kcal/mol), ZINC53298162 (?9.4 kcal/mol), ZINC70927268 (?10.0 kcal/mol), ZINC89878705 (?11.2 kcal/mol)) have shown better interaction with SmSirt2, in which ZINC89878705 (?11.2 kcal/mol) shows a more compact packing as compared to AKG2 and rest of ligands. These molecules could be further subject to in vitro study and model of SmSirt2 has been proposed for further structure-based drug design projects concerning sirtuins from Schistosoma mansoni.     

Two-dimensional 1H NMR studies of cytochrome c: hydrogen exchange in the N-terminal helix

The hydrogen exchange behavior of the N-terminal helical segment in horse heart cytochrome c was studied in both the reduced and the oxidized forms by use of two-dimensional nuclear magnetic resonance methods. The amide protons of the first six residues are not H bonded and exchange rapidly with solvent protons. The most N-terminal H-bonded groups--the amide NH of Lys-7 to Phe-10--exhibit a sharp gradient in exchange rate indicative of dynamic fraying behavior, consistent with statistical-mechanical principles. This occurs identically in both reduced and oxidized cytochrome c. In the oxidized form, residues 11-14, which form the last helical turn, all exchange with a similar rate, about one million times slower than the rate characteristic of freely exposed peptide NH, even though some are on the aqueous face of the helix and others are fully buried. These and similar observations in several other proteins appear to document local cooperative unfolding reactions as determinants of protein H exchange reactions. The N-terminal segment of cytochrome c is insensitive to the heme redox state, as in the crystallographic model, except for residues closest to the heme (Cys-14 and Ala-15), which exchange about 15-fold more slowly in the reduced form. The cytochrome c H exchange results can be further considered in terms of the conformation of the native and the transiently unfolded forms and their free energy relationships in both the reduced and the oxidized states.     

Syntheses, crystal structures, and hydrogen bonding patterns of 3'-C-methylenecarboxylic-3'-deoxythymidine and 3'-C-methyleneamidilylic-3'-deoxythymidine

Thymidine derivatives containing carboxylic acid and amide groups have been synthesized and the hydrogen-bonding patterns of 3'-C-methylenecarboxylic-3'-deoxythymidine 6 and 3'-C-methyleneamidilylic-3'-deoxythymidine 9 have been characterized by using X-ray crystallography.     

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: A case study in latarcins

In silico structural analyses of sets of α-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.     

Ca-ATPase structure in the E1 and E2 conformations: mechanism, helix-helix and helix-lipid interactions

The determination of the crystal structure of the Ca²⁺-ATPase of sarcoplasmic reticulum (SR) in its Ca²⁺-bound [Nature 405 (2000) 647] and Ca²⁺-free forms [Nature 418 (2002) 605] gives the opportunity for an analysis of conformational changes on the Ca²⁺-ATPase and of helix-helix and helix-lipid interactions in the transmembrane (TM) region of the ATPase. The locations of the ends of the TM α-helices on the cytoplasmic side of the membrane are reasonably well defined by the location of Trp residues and by the location of Lys-262 that snorkels up to the surface. The locations of the lumenal ends of the helices are less clear. The position of Lys-972 on the lumenal side of helix M9 suggests that the hydrophobic thickness of the protein is only about 21 Å, rather than the normal 30 Å. The experimentally determined TM α-helices do not agree well with those predicted theoretically. Charged headgroups are required for strong interaction of lipids with the ATPase, consistent with the large number of charged residues located close to the lipid-water interface. Helix packing appears to be rather irregular. Packing of helices M8 and M10 is of the 3-4 ridges-into-grooves or knobs-into-holes types. Packing of helices M5 and M7 involves two Gly residues in M7 and one Gly residue in M5. Packing of the other helices generally involves just one or two residues on each helix at the crossing point. The irregular packing of the TM α-helices in the Ca²⁺-ATPase, combined with the diffuse structure of the ATPase on the lumenal side of the membrane, is suggested to lead to a relative low activation energy for changing the packing of the TM α-helices, with changes in TM α-helical packing being important in the process of transfer of Ca²⁺ ions across the membrane. The inhibitor thapsigargin binds in a cleft between TM α-helices M3, M5 and M7. It is suggested that this and other similar clefts provide binding sites for a variety of hydrophobic molecules affecting the activity of the Ca²⁺-ATPase.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Structure-function analysis of tritrpticin analogs: potential relationships between antimicrobial activities, model membrane interactions, and their micelle-bound NMR structures

Tritrpticin is a member of the cathelicidin family of antimicrobial peptides. Starting from its native sequence (VRRFPWWWPFLRR), eight synthetic peptide analogs were studied to investigate the roles of specific residues in its biological and structural properties. This included amidation of the C-terminus paired with substitutions of its cationic and Phe residues, as well as the Pro residues that are important for its two-turn micelle-bound structure. These analogs were determined to have a significant antimicrobial potency. In contrast, two other peptide analogs, those with the three Trp residues substituted with either Phe or Tyr residues are not highly membrane perturbing, as determined by leakage and flip-flop assays using fluorescence spectroscopy. Nevertheless the Phe analog has a high activity; this suggests an intracellular mechanism for antimicrobial activity that may be part of the overall mechanism of action of native tritrpticin as a complement to membrane perturbation. NMR experiments of these two Trp-substituted peptides showed the presence of multiple conformers. The structures of the six remaining Trp-containing analogs bound to dodecylphosphocholine micelles showed major, well-defined conformations. These peptides are membrane disruptive and show a wide range in hemolytic activity. Their micelle-bound structures either retain the typical turn-turn structure of native tritrpticin or have an extended alpha-helix. This work demonstrates that closely related antimicrobial peptides can often have remarkably altered properties with complex influences on their biological activities.