Modulation of free energy landscapes as a strategy for the design of antimicrobial peptides

Computational design of antimicrobial peptides (AMPs) is a promising area of research for developing novel agents against drug-resistant bacteria. AMPs are present naturally in many organisms, from bacteria to humans, a time-tested mechanism that makes them attractive as effective antibiotics. Depending on the environment, AMPs can exhibit α-helical or β-sheet conformations, a mix of both, or lack secondary structure; they can be linear or cyclic. Prediction of their structures is challenging but critical for rational design. Promising AMP leads can be developed using essentially two approaches: traditional modeling of the physicochemical mechanisms that determine peptide behavior in aqueous and membrane environments and knowledge-based, e.g., machine learning (ML) techniques, that exploit ever-growing AMP databases. Here, we explore the conformational landscapes of two recently ML-designed AMPs, characterize the dependence of these landscapes on the medium conditions, and identify features in peptide and membrane landscapes that mediate protein-membrane association. For both peptides, we observe greater conformational diversity in an aqueous solvent than in a less polar solvent, and one peptide is seen to alter its conformation more dramatically than the other upon the change of solvent. Our results support the view that structural rearrangement in response to environmental changes is central to the mechanism of membrane-structure disruption by linear peptides. We expect that the design of AMPs by ML will benefit from the incorporation of peptide conformational substates as quantified here with molecular simulations.

     

Insights into the structure and molecular topography of the fatty acylated domain of synaptotagmin-1

Abundant attention has focused on synaptotagmin's C2 domains, but less is known about the structure and function of its other regions. Here, we synthesized the N-acetylated, C-end amidated and Cys-palmitated peptide (VLTCCFCICK KCLFKKKNKK K) which includes the fatty acylated cysteine residues in the membrane-affiliated domain of synaptotagmin-1. Fourier-transform infrared spectrometry indicated that this peptide's conformation is influenced by environmental polarity. In artificial bilayer membranes, this peptide exhibited abundant β-structure. Electron microscopy revealed that this peptide also promoted the stacking of liposome membranes. Together these results suggest that the fatty acylated region of synaptotagmin-1 is likely to adopt β-structure in biological membranes. This preference for β-structure versus α-helix has functional implications for the role of synaptotagmin-1 in synaptic vesicle exocytosis.     

Effect of salt on the interaction of Hal18 with lipid membranes

One of the major obstacles in the development of new antimicrobial peptides as novel antibiotics is salt sensitivity. Hal18, an α-helical subunit of Halocidin isolated from Halocynthia aurantium, has been previously shown to maintain its antimicrobial activity in high salt conditions. The α-helicity of Hal18 in the presence and absence of salt was demonstrated by circular dichroism spectroscopy, which showed that the peptide was mainly unordered containing β-strands and β-turns. However, in the presence of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS) vesicles, Hal18 folded to form α-helices (circa 42?%). Furthermore, the structure was not significantly affected by pH or the presence of metal ions. These data were supported by monolayer results showing Hal18 induced stable surface pressure changes in monolayers composed of DMPC (5?mN?m(-1)) and DMPS (8.5?mN?m(-1)), which again were not effected by the presence of metal ions or pH. It is proposed that the hydrophobic groove within its molecular architecture enables the peptide to form stable associations with lipid membranes. The balance of hydrophobicity along the Hal18 long axis would also support oblique orientation of the peptide at the membrane interface. Hence, this model of membrane interaction would enable the peptide to penetrate deep into the membrane. This concept is supported by lysis data. Overall, it would appear that this peptide is a potential candidate for future AMP design for use in high salt environments.     

Synthesis and antimicrobial activity against Pseudomonas aeruginosa of macrocyclic β-hairpin peptidomimetic antibiotics containing N-methylated amino acids

Antimicrobial resistance among Gram-negative bacteria is a growing problem, fueled by the paucity of new antibiotics that target these microorganisms. One novel family of macrocyclic β-hairpin-shaped peptidomimetics was recently shown to act specifically against Pseudomonas spp. by a novel mechanism of action, targeting the outer membrane protein LptD, which mediates lipopolysaccharide transport to the cell surface during outer membrane biogenesis. Here we explore the mode of binding of one of these β-hairpin peptidomimetics to LptD in Pseudomonas aeruginosa, by examining the effects on antimicrobial activity following N-methylation of individual peptide bonds. An N-methyl scan of the cyclic peptide revealed that residues on both sides of the β-hairpin structure at a non-hydrogen bonding position likely mediate hydrogen-bonding interactions with the target LptD. Structural analyses by NMR spectroscopy further reinforce the conclusion that the folded β-hairpin structure of the peptidomimetic is critical for binding to the target LptD. Finally, new NMe analogues with potent activity have been identified, which opens new avenues for optimization in this family of antimicrobial peptides.     

Hipposin,a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)

A novel 51-residue antimicrobial peptide (AMP) from the skin mucus of Atlantic halibut (Hippoglossus hippoglossus L.) was isolated using acid extraction, and cationic exchange and reversed phase chromatography. The complete amino acid sequence of the AMP, termed hipposin, was determined by automated Edman degradation and mass spectrometry to be SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAHRVGAGAPVYL. The N-terminal amino group was acetylated. The theoretical mass of hipposin was calculated to be 5458.4 Da, which was in good agreement with the mass of 5459 Da determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Hipposin was shown to be derived from histone H2A by PCR amplifying the encoding sequences from Atlantic halibut genomic DNA. The peptide showed sequence similarity with the 39-mer AMP buforin I of Asian toad and the 19-mer AMP parasin I of catfish. Fifty of the fifty-one residues in hipposin were identical to the N-terminal region of histone H2A from rainbow trout. Hipposin showed strong antimicrobial activity against several Gram-positive and Gram-negative bacteria and activity could be detected down to hipposin concentrations of 0.3 microM (1.6 microg/ml). Hipposin without N-terminal acetylation was prepared by solid-phase peptide synthesis and shown to have the same antimicrobial activity as the natural acetylated peptide. Thus, hipposin is a new broad-spectrum histone-derived AMP found in the skin mucus of Atlantic halibut.     

Antimicrobial activity and structure of a consensus human β-defensin and its comparison to a novel putative hBD10

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human β-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical β-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in ¹H NMR spectra and structural studies were not pursued. The evaluation of different β-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.     

Structure and antimicrobial activity of platypus ‘intermediate’ defensin-like peptide

The three-dimensional structure of a chemically synthesized peptide that we have called ‘intermediate’ defensin-like peptide (Int-DLP), from the platypus genome, was determined by nuclear magnetic resonance (NMR) spectroscopy; and its antimicrobial activity was investigated. The overall structural fold of Int-DLP was similar to that of the DLPs and β-defensins, however the presence of a third antiparallel β-strand makes its structure more similar to the β-defensins than the DLPs. Int-DLP displayed potent antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. The four arginine residues at the N-terminus of Int-DLP did not affect the overall fold, but were important for its antimicrobial potency.     

Distinct mode of membrane interaction and disintegration by diverse class of antimicrobial peptides

The exploitation of conventional antibiotics in conjunction with the adeptness of microbes has led to the emergence of multi-drug-resistant pathogens. This has posed a severe threat to combating life-threatening infectious diseases. Antimicrobial peptides (AMP), which are considered to be the first line of defense in all living organisms, are being developed for therapeutic use. Herein, we determined the NMR solution structure of Rhesus macaque Myeloid Alpha Defensin-4 (RMAD4), a defensin AMP. Additionally, the distinct modes of membrane perturbation for two structurally dissimilar classes of AMPs was studied using biophysical methods namely, Solid-state ³¹P NMR, DSC and cryo-TEM. The cathelicidin - Bovine myeloid antimicrobial peptide (BMAP-28 (1–18)), which adopts a helical conformation, and the defensin RMAD4 peptide that natively folds to form β-sheets appeared to engage differently with the bacterial membrane. The helical BMAP-28 (1–18) peptide initiates lipid segregation and membrane thinning followed by pore formation, while the β-stranded RMAD4 peptide demonstrates fragmentation of the bilayer by the carpet or detergent-like mechanism of action. Molecular dynamics studies sufficiently corroborated these findings. The structure and mechanism of action of the AMPs studied using experimental and computational approaches are believed to help in providing a platform for the rational design of new competent and cost-effective antimicrobial peptides for therapeutic applications.     

Design and production of a novel antimicrobial fusion protein in Escherichia coli

In recent years, antimicrobial peptides (AMPs) have attracted increasing attention. The microbial cells provide a simple, cost-effective platform to produce AMPs in industrial quantities. While AMP production as fusion proteins in microorganisms is commonly used, the recovery of AMPs necessitates the use of expensive proteases and extra purification steps. Here, we develop a novel fusion protein DAMP4-F-pexiganan comprising a carrier protein DAMP4 linked to the AMP, pexiganan, through a long, flexible linker. We show that this fusion protein can be purified using a non-chromatography approach and exhibits the same antimicrobial activity as the chemically synthesized pexiganan peptide without any cleavage step. Activity of the fusion protein is dependent on a long, flexible linker between the AMP and carrier domains, as well as on the expression conditions of the fusion protein, with low-temperature expression promoting better folding of the AMP domain. The production of DAMP4-F-pexiganan circumvents the time-consuming and costly steps of chromatography-based purification and enzymatic cleavages, therefore shows considerable advantages over traditional microbial production of AMPs. We expect this novel fusion protein, and the studies on the effect of linker and expression conditions on its antimicrobial activity, will broaden the rational design and production of antimicrobial products based on AMPs.

     

Molecular Characterisation and Phylogenetic Analysis of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Zc-hepc1), from the Coral Fish Moorish idol, Zanclus cornutus (Linnaeus, 1758)

Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of +1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a β-hairpin-like structure with two antiparallel β-sheets. This is the first report of an AMP from the coral fish Z. cornutus.     

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.     

Trichoplaxin — A new membrane-active antimicrobial peptide from placozoan cDNA

A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.     

EcDBS1R6: A novel cationic antimicrobial peptide derived from a signal peptide sequence

BackgroundBacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm.MethodsAntimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure.ResultsSignal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide.ConclusionThe strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents.General significanceThe process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.     

Peptidomics-based discovery of an antimicrobial peptide derived from insulin-like growth factor-binding protein 5

Antimicrobial peptides (AMPs) are effector molecules that are able to kill or inactivate microbial pathogens. However, most AMPs harbor multiple basic amino acids that hamper current proteomic identification. In our peptidomic survey of endogenous peptides, we identified a novel intramolecular disulfide-linked 22-residue amidated peptide. This peptide, designated AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5), showed antimicrobial activity against six of the eight microorganisms tested at concentrations comparable to or lower than those for well-characterized AMPs cathelicidin and β-defensin-2. AMP-IBP5 is identical at the amino acid level between human, mouse, rat, pig, and cow. Natural occurrence of this peptide as the originally isolated form was demonstrated in the rat brain and intestine, using mass spectrometric characterization of major immunoreactivity. The peptide is flanked N-terminally by a single arginine and C-terminally by a common amidation signal, indicating that insulin-like growth factor-binding protein 5 (IGFBP-5) undergoes specific cleavage by a defined set of processing proteases. Furthermore, the intramolecular linkage C199-C210 reveals itself as a correct disulfide pairing in the precursor protein, the finding not inferred from closely related family members IGFBP-4 and -6. In principle, neither conventional proteomics nor bioinformatics would achieve the identification of this AMP. Our study exemplifies the impact of peptidomics to study naturally occurring peptides.     

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.     

A beta-defensin 1-like antimicrobial peptide from the tree shrew, Tupaia belangeri

A novel beta-defensin 1-like antimicrobial peptide (β-defensin 1TB) containing 36 amino acid residues was purified and characterized from the serum of the tree shrew, Tupaia belangeri. Its amino acid sequence was determined as DHYLCVKNEGICLYSSCPSYTKIEGTCYGGKAKCCK, by Edman degradation, mass spectrometry analysis, and cDNA cloning. Evolution analysis indicated that β-defensin 1TB showed maximal similarity to the β-defensin 1 identified from cotton-top tamarin, Saguinus oedipus. β-defensin 1TB exerted potential antimicrobial activities against wide spectrum of microorganisms including Gram-negative and -positive bacteria and fungi. It showed little hemolitic activity to human or rabbit red cells. To the best of our knowledge, this is the first report of antimicrobial peptide from Tupaiidae.     

Potentiometric titration of poly-L-lysine: the coil-to-beta transition

We have performed potentiometric titrations of poly-L -lysine. From these data we have calculated the free energy and enthalpy changes for the folding of the random coil to the α-helix in 10% ethanol (?120 and ?120 cal/mole) and from the random coil to the β-structure in water (?140 and 870 cal/mole) and in 10% ethanol (?180 and 980 cal mole). Comparison of these values with each other and with values for the coil → α- helix transition in water (?78 and ?880 cal/mole) led to the following conclusions. The stabilization by ethanol of ethanol of the α-helix with respect to the coil is that predicted from the known free energy of transfer of the peptide group from water to 10% ethanol. Similar data to explain the enthalpy difference are not available. The thermodynamic functions for the transition from α-helix to β-structure, obtained by subtracting those for the coil → α-helix and coil → β-structure transitions, are explained from a consideration of the structural differences: non bonded interactions of the polypeptide backbone are less favorable in the β-structure than in the α-helix, causing an increase in the energy, while hydrophobic contacts between side chains raise the entropy of the β-structure as compared with the α-helix, so that the free energy difference between the two structures is small, but enthalpy and entropy differences are large. The observation of only small differences in the free energy and enthalpy changes for the transition from coil β-structure upon going from water to 10% ethanol is expected by considering both the free energy of transfer of the peptide group (as for the α-helix) and the free energy and enthalpy of transfer of the apolar part of the side chain involved in hydrophobic bond formation.     

Conformational behavior of the HAV-VP3(110–121) peptidic sequence and synthetic analogs in membrane environments studied by CD and computational methods

The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110–121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic β-structure of the peptide. To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the β-structure in the presence of liposomes. © 1998 John Wiley & Sons, Inc. Biopoly 45: 479–492, 1998     

A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056

The novel antimicrobial peptide with a dimeric dendrimer scaffold, SB056, was empirically optimized by high-throughput screening. This procedure produced an intriguing primary sequence whose structure-function analysis is described here. The alternating pattern of hydrophilic and hydrophobic amino acids suggests the possibility that SB056 is a membrane-active peptide that forms amphiphilic β-strands in a lipid environment. Circular dichroism confirmed that the cationic SB056 folds as β-sheets in the presence of anionic vesicles. Lipid monolayer surface pressure experiments revealed unusual kinetics of monolayer penetration, which suggest lipid-induced aggregation as a membranolytic mechanism. NMR analyses of the linear monomer and the dendrimeric SB056 in water and in 30% trifluoroethanol, on the other hand, yielded essentially unstructured conformations, supporting the excellent solubility and storage properties of this compound. However, simulated annealing showed that many residues lie in the β-region of the Ramachandran plot, and molecular-dynamics simulations confirmed the propensity of this peptide to fold as a β-type conformation. The excellent solubility in water and the lipid-induced oligomerization characteristics of this peptide thus shed light on its mechanism of antimicrobial action, which may also be relevant for systems that can form toxic β-amyloid fibrils when in contact with cellular membranes. Functionally, SB056 showed high activity against Gram-negative bacteria and some limited activity against Gram-positive bacteria. Its potency against Gram-negative strains was comparable (on a molar basis) to that of colistin and polymyxin B, with an even broader spectrum of activity than numerous other reference compounds.     

基于BERT与Text-CNN的抗菌肽识别方法

抗菌肽(antimicrobial peptides,AMPs)广泛存在于生命体中,是一种具有广谱抗菌活性、免疫调节功能的小分子多肽。抗菌肽不易产生耐药性,适用范围广,具有极大的临床价值,是传统抗生素的有力竞争者。识别抗菌肽是抗菌肽研究领域中的重要研究方向,湿实验法在进行大规模抗菌肽识别时存在成本高、效率低、周期长等难点,计算机辅助识别法是抗菌肽识别手段的重要补充,如何提升准确率是其中的关键问题。蛋白质序列可以被近似地看作是由氨基酸组成的语言,运用自然语言处理(natural language processing,NLP)技术可能提取到丰富的特征。本文将自然语言处理领域中的预训练模型BERT和微调结构Text-CNN结合,对蛋白质语言进行建模,提供了开源可用的抗菌肽识别工具,并与已发表的5种抗菌肽识别工具进行了比较。结果表明,优化“预训练-微调”策略带来了准确率、敏感度、特异性和马修相关系数的整体提升,为进一步研究抗菌肽识别算法提供了新思路。