Molecular dynamics studies of the antimicrobial peptides piscidin 1 and its mutants with a DOPC lipid bilayer

Piscidin 1 (Pis‐1) has a high broad‐spectrum activity against bacteria, fungi, and viruses but it also has a moderate hemolytic activities. To improve the antibacterial activity and to reduce toxicity, mutants Pis‐1AA (G8A/G13A double mutant) and Pis‐1PG (G8P mutant) have been designed based on the crystal structure of Pis‐1. Eighteen independent molecular dynamics (MD) simulations of Pis‐1 and its mutants with membranes are conducted in this article. Furthermore, 60 independent MD simulations of three peptides in water box have also been discussed for comparison. The results indicate that the unfolding process starts at the middle of the peptide. Pis‐1 disrupts easily in the region of Val10‐Lys14. Pis‐1PG has a flexible N‐terminal region, and the interaction between N‐terminal and C‐terminal is very weak. Pis‐1AA has the most stable helical structure. In addition, percentage of native contacts and hydrogen bonds analysis are also performed. Lipid‐peptide interaction analysis suggests that Pis‐1 and Pis‐1AA has a stronger interaction with the zwitterionic dioleoylphosphatidylcholine (DOPC) lipid bilayer than Pis‐1PG. When compared with the results of peptide with membrane, peptides are unstable and unfolding quickly in water solution. Our results are applicable in examining diversities on hemolytic, antibacterial, and selectivity of antimicrobial peptides. © 2012 Wiley Periodicals, Inc. Biopolymers 97:998–1009, 2012.     

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.     

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.     

Effects of the peptide Magainin H2 on Supported Lipid Bilayers studied by different biophysical techniques

Given the increasing trend in bacterial antibiotic resistance, research on antimicrobial peptides and their mechanisms of action has become of huge relevance in the last years. Several studies have investigated the effects of a large variety of antimicrobial peptides directly on bacteria or on model lipid bilayers. In the case of model lipid bilayers, different systems are typically exploited; however, different results could be obtained due to the specific properties of the used system. Supported Lipid Bilayers and Giant Unilamellar Vesicles are among the most popular model systems. Here we used Atomic Force Microscopy and fluorescence microscopy to study the interaction of the antimicrobial peptide Magainin H2, an analog of Magainin 2 with increased hydrophobicity, on Supported Lipid Bilayers. We found that, for this kind of model bilayer, due to its strong interaction with the support, the lateral expansion of the membrane induced by the interaction with the peptides is initially inhibited and subsequently proceeds creating new bilayer regions with many defects. This scenario gives rise in Supported Lipid Bilayers to effects like initial increase of lateral pressure, formation of lipid tubes to release this increase, or development of bilayer regions with lower lipid density. Our results highlight that care should be given to the selected model system when studying and comparing the interaction of peptides with other lipid bilayer model systems.     

Coupling Molecular Dynamics Simulations with Experiments for the Rational Design of Indolicidin-Analogous Antimicrobial Peptides

Antimicrobial peptides (AMPs) have attracted much interest in recent years because of their potential use as new-generation antibiotics. Indolicidin (IL) is a 13-residue cationic AMP that is effective against a broad spectrum of bacteria, fungi, and even viruses. Unfortunately, its high hemolytic activity retards its clinical applications. In this study, we adopted molecular dynamics (MD) simulations as an aid toward the rational design of IL analogues exhibiting high antimicrobial activity but low hemolysis. We employed long-timescale, multi-trajectory all-atom MD simulations to investigate the interactions of the peptide IL with model membranes. The lipid bilayer formed by the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was chosen as the model erythrocyte membrane; lipid bilayers formed from a mixture of POPC and the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were chosen to model bacterial membranes. MD simulations with a total simulation time of up to 4 μs revealed the mechanisms of the processes of IL adsorption onto and insertion into the membranes. The packing order of these lipid bilayers presumably correlated to the membrane stability upon IL adsorption and insertion. We used the degree of local membrane thinning and the reduction in the order parameter of the acyl chains of the lipids to characterize the membrane stability. The order of the mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/POPC lipid bilayer reduced significantly upon the adsorption of IL. On the other hand, although the order of the pure-POPC lipid bilayer was perturbed slightly during the adsorption stage, the value was reduced more dramatically upon the insertion of IL into the membrane's hydrophobic region. The results imply that enhancing IL adsorption on the microbial membrane may amplify its antimicrobial activity, while the degree of hemolysis may be reduced through inhibition of IL insertion into the hydrophobic region of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7 → Lys), IL-F89 (Trp8 and Trp9 → Phe), and IL-K7F89 (Pro7 → Lys; Trp8 and Trp9 → Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach—coupling MD simulations with experimental design—is a useful strategy toward the rational design of AMPs for potential therapeutic use.     

Molecular dynamics simulations of pentapeptides at interfaces: salt bridge and cation-pi interactions

Peptide-membrane interactions are important for understanding the binding, partitioning, and folding of membrane proteins; the activity of antimicrobial and fusion peptides; and a number of other processes. We describe molecular dynamics simulations (10-25 ns) of two pentapeptides Ace-WLXLL (with X = Arg or Lys side chain) (White, S. H., and Wimley, W.C. (1996) Nat. Struct. Biol. 3, 842-848) in water and three different membrane mimetic systems: (i) a water/cyclohexane interface, (ii) water-saturated octanol, and (iii) a solvated dioleoylphosphatidylcholine bilayer. A salt bridge is found between the protonated Arg or Lys side chains with the carboxyl terminus at the three interfaces. In water/cyclohexane, the salt bridge is most exposed to the water phase and least stable. In water/octanol and the lipid bilayer systems, the salt bridge once formed persists throughout the simulations. In the lipid bilayer, the salt bridge is more stable when the peptide penetrates deeper into the bilayer. In one of two peptides, a cation-pi interaction between the Arg and the Trp side chains is stable in the lipid bilayer for about 15 ns before breaking. In all cases, the conformations of the peptides are restricted by their presence at the interface and can be assigned to a few major conformational clusters. Side chains facing the water phase are most mobile. In the lipid bilayer, the peptides remain in the interface area, where they overlap with the carbonyl area of the lipid bilayer and perturb the local density profile of the bilayer. The tryptophan side chain remains in the water-lipid interface, where it interacts with the lipid choline group and forms hydrogen bonds with the ester carbonyl of the lipid and with water in the interface.     

Simulation studies of the interaction of antimicrobial peptides and lipid bilayers

Experimental studies of a number of antimicrobial peptides are sufficiently detailed to allow computer simulations to make a significant contribution to understanding their mechanisms of action at an atomic level. In this review we focus on simulation studies of alamethicin, melittin, dermaseptin and related antimicrobial, membrane-active peptides. All of these peptides form amphipathic alpha-helices. Simulations allow us to explore the interactions of such peptides with lipid bilayers, and to understand the effects of such interactions on the conformational dynamics of the peptides. Mean field methods employ an empirical energy function, such as a simple hydrophobicity potential, to provide an approximation to the membrane. Mean field approaches allow us to predict the optimal orientation of a peptide helix relative to a bilayer. Molecular dynamics simulations that include an atomistic model of the bilayer and surrounding solvent provide a more detailed insight into peptide-bilayer interactions. In the case of alamethicin, all-atom simulations have allowed us to explore several steps along the route from binding to the membrane surface to formation of transbilayer ion channels. For those antimicrobial peptides such as dermaseptin which prefer to remain at the surface of a bilayer, molecular dynamics simulations allow us to explore the favourable interactions between the peptide helix sidechains and the phospholipid headgroups.     

Position and orientation of gallated proanthocyanidins in lipid bilayer membranes: influence of polymerization degree and linkage type

It is well known that the biological activity of gallated proanthocyanidins (PAs) is highly structure-dependent. Polymerization degree (DP) and linkage types affect their biological activity greatly. Positions and orientations of gallated PAs in lipid bilayer reveal their structure-function activity at the molecular level. The present work aimed at determining the locations and orientations of epigallocatechin-3-gallate (EGCG) and its derivatives: A-type and B-type EGCG dimers and trimers in 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) lipid bilayer via molecular dynamic (MD) simulations. The results showed that EGCG and its derivatives localized in the lipid bilayer or on the bilayer/water interface. Their penetration depths and orientations depended on both DP and linkage types. The penetration depths decreased with the increase of DP, sequencing to be EGCG > EGCG dimers > EGCG trimers. Spatially stretched A-type PAs could form more hydrogen bonds (H-bonds) with deep oxygen atoms of lipid bilayer and have higher affinity to the lipid bilayer than B-type PAs. Our results will provide an explicit evidence for PAs’ distinct biological activities.     

Structure and dynamics of cationic membrane peptides and proteins: insights from solid-state NMR

Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein-lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides.     

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.     

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.     

Differential scanning calorimetry and X-ray diffraction studies of the specificity of the interaction of antimicrobial peptides with membrane-mimetic systems

Interest in biophysical studies on the interaction of antimicrobial peptides and lipids has strongly increased because of the rapid emergence of antibiotic-resistant bacterial strains. An understanding of the molecular mechanism(s) of membrane perturbation by these peptides will allow a design of novel peptide antibiotics as an alternative to conventional antibiotics. Differential scanning calorimetry and X-ray diffraction studies have yielded a wealth of quantitative information on the effects of antimicrobial peptides on membrane structure as well as on peptide location. These studies clearly demonstrated that antimicrobial peptides show preferential interaction with specific phospholipid classes. Furthermore, they revealed that in addition to charge-charge interactions, membrane curvature strain and hydrophobic mismatch between peptides and lipids are important parameters in determining the mechanism of membrane perturbation. Hence, depending on the molecular properties of both lipid and peptide, creation of bilayer defects such as phase separation or membrane thinning, pore formation, promotion of nonlamellar lipid structures or bilayer disruption by the carpet model or detergent-like action, may occur. Moreover, these studies suggest that these different processes may represent gradual steps of membrane perturbation. A better understanding of the mutual dependence of these parameters will help to elucidate the molecular mechanism of membrane damage by antimicrobial peptides and their target membrane specificity, keys for the rationale design of novel types of peptide antibiotics.     

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from ¹⁵N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are α-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. ¹⁵N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.     

Binding and insertion of alpha-helical anti-microbial peptides in POPC bilayers studied by molecular dynamics simulations

We have performed molecular dynamics simulations of the interactions of two alpha-helical anti-microbial peptides, magainin2 and its synthetic analog of MSI-78, with palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers. We used various initial positions and orientations of the peptide with respect to the lipid bilayer, including a surface-bound state parallel to the interface, a trans-membrane state, and a partially inserted state. Our 20 ns long simulations show that both magainin2 and MSI-78 are most stable in the lipid environment, with the peptide destabilized to different extents in both aqueous and lipid/water interfacial environments. We found that there are strong specific interactions between the lysine residues of the peptides and the lipid head-group regions. MSI-78, owing to its large number of lysines, shows better binding characteristics and overall stability when compared to magainin2. We also find that both peptides destabilize the bilayer environment, as observed by the increase in lipid tail disorder and the induction of local curvature on the lipid head-groups by the peptides. From all the simulations, we conclude that the hydrogen bonding interactions between the lysines of the peptides and the oxygens of the polar lipid head-groups are the strongest and determine the overall peptide binding characteristics to the lipids.     

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.     

Folding profiles of antimicrobial scorpion venom-derived peptides on hydrophobic surfaces: a molecular dynamics study

Abstract

Most helical antimicrobial peptides (AMPs) are usually unfolded in aqueous solution; however they acquire their secondary structure in the presence of a hydrophobic environment such as lipid membranes. Being the biological membranes the main target of many AMPs it is necessary to understand their way of action. Pandinin 2 (Pin2) is an alpha-helical AMP isolated from the venom of the African scorpion Pandinus imperator which shows high antimicrobial activity against Gram-positive bacteria and it is less active against Gram-negative bacteria, nevertheless, it has strong hemolytic activity. Its chemically synthesized Pin2GVG analog has low hemolytic activity while keeping its antimicrobial activity. With the aim of exploring the partition and subsequent folding of these peptides, in this work we report the results of extensive molecular dynamics simulations of Pin2 and Pin2GVG peptides in the presence of 2 hydrophobic environments such as dodecyl-phosphocholine (DPC) micelle and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) membrane. Our results indicate that Pin2 folds in DPC with a 79% of alpha-helical content, which is in agreement with the experimental results, while in POPC it has 62.5% of alpha-helical content. On the other hand, Pin2GVG presents a higher percentage of alpha-helical structure in POPC and a smaller content in DPC when compared with Pin2. These results can help to better choose the starting structures in future molecular dynamics simulations of AMPs, because these peptides can adopt slightly different conformations depending on the hydrophobic environment.

Communicated by Ramaswamy H. Sarma     

Molecular dynamics simulation of neuropeptide B and neuropeptide W in the dipalmitoylphosphatidylcholine membrane bilayer

GPR7 and GPR8 are recently deorphanized G-protein-coupled receptors that are implicated in the regulation of neuroendocrine function, feeding behavior, and energy homeostasis. Neuropeptide B (NPB) and neuropeptide W (NPW) are two membrane-bound hypothalamic peptides, which specifically antagonize GPR7 and GPR8. Despite years of research, an accurate estimation of structure and molecular recognition of these neuropeptide systems still remains elusive. Herein, we investigated the structure, orientation, and interaction of NPB and NPW in a dipalmitoylphosphatidylcholine bilayer using long-range molecular dynamics (MD) simulation. During 30-ns simulation, membrane-embedded helical axes of NPB and NPW tilted 30 and 15°, respectively, from the membrane normal in order to overcome possible hydrophobic mismatch with the lipid bilayer. The calculation of various structural parameters indicated that NPW is more rigid and compact as compared to NPB. Qualitatively, the peptides exhibited flexible N-terminal (residues 1–12) and rigid C-terminal α-helical parts (residues 13–21), confirming previous NMR data. A strong electrostatic attraction between C-termini and headgroup atoms caused translocation of the peptides towards lower leaflet of the bilayer. The stabilizing hydrogen bonds (H-bonds) between phosphate groups and Trp1, Lys3, and Arg15 of the peptides played important roles for membrane anchoring. MD simulations of Alanine (Ala) mutants revealed that WYK->Ala variant of NPB/NPW lacked crucial H-bond interactions with phospholipid headgroups and also caused severe misfolding in NPB. Altogether, the knowledge of preferred structural fold and interaction of neuropeptides within the membrane bilayer will be useful to develop synthetic agonist or antagonist peptides for GPR7 and GPR8.     

Interactions of alpha-helices with lipid bilayers: a review of simulation studies

Membrane proteins, of which the majority seem to contain one or more alpha-helix, constitute approx. 30% of most genomes. A complete understanding of the nature of helix/bilayer interactions is necessary for an understanding of the structural principles underlying membrane proteins. This review describes computer simulation studies of helix/bilayer interactions. Key experimental studies of the interactions of alpha-helices and lipid bilayers are briefly reviewed. Surface associated helices are found in some membrane-bound enzymes (e.g. prostaglandin synthase), and as stages in the mechanisms of antimicrobial peptides and of pore-forming bacterial toxins. Transmembrane alpha-helices are found in most integral membrane proteins, and also in channels formed by amphipathic peptides or by bacterial toxins. Mean field simulations, in which the lipid bilayer is approximated as a hydrophobic continuum, have been used in studies of membrane-active peptides (e.g. alamethicin, melittin, magainin and dermaseptin) and of simple membrane proteins (e.g. phage Pf1 coat protein). All atom molecular dynamics simulations of fully solvated bilayers with transmembrane helices have been applied to: the constituent helices of bacteriorhodopsin; peptide-16 (a simple model TM helix); and a number of pore-lining helices from ion channels. Surface associated helices (e.g. melittin and dermaseptin) have been simulated, as have alpha-helical bundles such as bacteriorhodopsin and alamethicin. From comparison of the results from the two classes of simulation, it emerges that a major theoretical challenge is to exploit the results of all atom simulations in order to improve the mean field approach.     

A Peptide Derived from LFA-1 Protein that Modulates T-cell Adhesion Binds to Soluble ICAM-1 Protein

Abstract

Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM- 1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM- 1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using “autodock” resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.