Solution structure of LCI, a novel antimicrobial peptide from Bacillus subtilis

LCI, a 47-residue cationic antimicrobial peptide (AMP) found in Bacillus subtilis, is one of the main effective components that have strong antimicrobial activity against Xanthomonas campestris pv Oryzea and Pseudomonas solanacearum PE1, etc. To provide insight into the activity of the peptide, we used nuclear magnetic resonance spectroscopy to determine the structure of recombinant LCI. The solution structure of LCI has a novel topology, containing a four-strand antiparallel β-sheet as the dominant secondary structure. It is the first structure of the LCI protein family. Different from any known β-structure AMPs, LCI contains no disulfide bridge or circular structure, suggesting that LCI is also a novel β-structure AMP.     

Solution structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide

Penaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen-3a analogue was determined by using two-dimensional 1H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29-58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys32-Cys47 and Cys48-Cys55). These two coils are in turn linked together by the third disulfide bond (Cys36-Cys54). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg45 and Arg50, which belong to the helix, and side chains of Arg37 and Arg53, which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences.     

Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.     

Solution NMR structure of a model class A (apolipoprotein) amphipathic alpha helical peptide

To better understand the structural determinants of the physical-chemical and the biological properties of Ac-18A-NH(2) (acetyl-AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe-amide), we have determined its structure in 50% (v/v) trifluroethanol (TFE-d(3))/water mixture (5 mM potassium phosphate, pH 5.5, 310K) using two-dimensional proton NMR spectroscopy. Stereospecific assignments have been made for C(beta)H protons (all the residues except Ala and Val) and gammaCH(3) (Val) groups. Nuclear Overhauser effects are observed between the nonpolar side chains spaced at (i) and (i + 4) position in the primary sequence, e.g., Trp2 and Phe6, and Phe6 and Val10. This suggests that in addition to N-terminal acetyl and C-terminal amide groups, the amphipathic alpha helical structure of Ac-18A-NH(2) is further stabilized by interactions between the hydrophobic residues on the nonpolar face of the helix.     

两种合成抗菌肽的结构及抗菌作用机理

为深入了解两种新型人工抗菌肽mytilin-derived-peptide-1 (MDP-1) 和mytilin-derived-peptide-2 (MDP-2) 的溶液结构和抗菌机理并探讨两种抗菌肽之间活性差异的结构基础,采用二维核磁共振技术 (2-D NMR) 研究MDP分子的溶液结构;采用透射电镜技术 (Transmitted electron microscopy,TEM) 研究MDP分子对于大肠杆菌和藤黄叠球菌的作用机理。研究结果表明,MDP-1和MDP-2均采取了典型的β-发夹结构,其分子表面具有明显的疏水斑片,其分子中碱性氨基酸突出于分子表面;经MDP分子处理后的大肠杆菌以及藤黄叠球菌均出现细胞壁或细胞膜结构被破坏,并出现膜壁分离以及细胞质内缩现象。我们认为,MDP-1和MDP-2分子中的碱性氨基酸有助于MDP结合细菌表面的带负电荷的基团,同时其分子表面的疏水斑片有助于其插入到细菌细胞膜内;其疏水斑片面积以及碱性氨基酸在分子表面的拓扑结构差异是MDP-1和MDP-2活性差异的主要原因;电镜实验结果表明MDP-1和MDP-2的主要靶标是细菌细胞壁以及细胞膜;上述研究为深入了解MDP分子的结构与功能的关系以及将来基于MDP分子的药物研发奠定了基础。     

Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy.

CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram-positive and gram-negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly alpha-helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic alpha-helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic alpha-helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Studies of synthetic peptide analogs of the amphipathic helix. Correlation of structure with function

The four peptide analogs of the amphipathic helix whose interactions with dimyristoyl phosphatidylcholine were described in the preceding paper were compared with apolipoproteins (apo) A-I and A-II in ability to displace native apolipoprotein from high density lipoprotein (HDL) and in ability to activate lecithin:cholesterol acyltransferase. The rank order of the ability of the four peptide analogs to displace apo-A-I from intact HDL was 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, the same order suggested in the preceding paper for relative lipid affinities. Modified HDL from which 40% of the apo-A-I had been displaced by 18A was indistinguishable from unmodified HDL in its ability to act as a lecithin:cholesterol acyltransferase substrate. This suggests that the easily displaced apo-A-I molecules in polydisperse HDL are relatively ineffectual as lecithin:cholesterol acyltransferase activators and/or 18A replaces the lecithin:cholesterol acyltransferase activity lost. The peptide analog 18A-Pro-18A was found to be a powerful activator of lecithin:cholesterol acyltransferase when incubated with unilamellar egg phosphatidylcholine (PC) vesicles, reaching 140% of the activity of apo-A-I at a 1:1.75 peptide-to-egg PC ratio. In another experiment, it was found that discoidal egg PC complexes of 18A-Pro-18A, 18A, and des-Val10-18A, formed by cholate dialysis, had 30-45% of the activity of apo-A-I/egg PC discoidal complexes, also formed by cholate dialysis, at the same peptide/lipid weight ratio. Examination of the structures formed when the 18A-Pro-18A peptide was incubated with unilamellar egg PC vesicles indicated that the ability of 18A-Pro-18A to exceed apo-A-I in lecithin:cholesterol acyltransferase activating ability is due to the spontaneous conversion by 18A-Pro-18A of egg PC vesicles to small protein annulus-bilayer disc structures. Apo-A-I, apo-A-II, nor any of the other three peptide analogs of the amphipathic helix studied were able to convert a significant fraction of egg PC unilamellar vesicles to discoidal structures.     

Solution structure of termicin,an antimicrobial peptide from the termite Pseudacanthotermes spiniger

The solution structure of termicin from hemocytes of the termite Pseudacanthotermes spiniger was determined by proton two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling techniques. Termicin is a cysteine-rich antifungal peptide also exhibiting a weak antibacterial activity. The global fold of termicin consists of an alpha-helical segment (Phe4-Gln14) and a two-stranded (Phe19-Asp25 and Gln28-Phe33) antiparallel beta-sheet forming a "cysteine stabilized alphabeta motif" (CSalphabeta) also found in antibacterial and antifungal defensins from insects and from plants. Interestingly, termicin shares more structural similarities with the antibacterial insect defensins and with MGD-1, a mussel defensin, than with the insect antifungal defensins such as drosomycin and heliomicin. These structural comparisons suggest that global fold alone does not explain the difference between antifungals and antibacterials. The antifungal properties of termicin may be related to its marked hydrophobicity and its amphipatic structure as compared to the antibacterial defensins. [SWISS-PROT accession number: Termicin (P82321); PDB accession number: 1MM0.]     

Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana

Antimicrobial peptides are important defense compounds of higher organisms that can be used as therapeutic agents against bacterial and/or viral infections. We designed several antimicrobial peptides containing hydrophobic and positively charged clusters that are active against plant and human pathogens. Especially peptide SP1-1 is highly active with a MIC value of 0.1 μg/ml against Xanthomonas vesicatoria, Pseudomonas corrugata and Pseudomonas syringae pv syringae. However, for commercial applications high amounts of peptide are necessary. The synthetic production of peptides is still quite expensive and, depending on the physico-chemical features, difficult. Therefore we developed a plant/tobacco mosaic virus-based production system following the ‘full virus vector strategy’ with the viral coat protein as fusion partner for the designed antimicrobial peptide. Infection of Nicotiana benthamiana plants with such recombinant virus resulted in production of huge amounts of virus particles presenting the peptides all over their surface. After extraction of recombinant virions, peptides were released from the coat protein by chemical cleavage. A protocol for purification of the antimicrobial peptides using high resolution chromatographic methods has been established. Finally, we yielded up to 0.025 mg of peptide per g of infected leaf biomass. Mass spectrometric and NMR analysis revealed that the in planta produced peptide differs from the synthetic version only in missing of N-terminal amidation. But its antimicrobial activity was in the range of the synthetic one. Taken together, we developed a protocol for plant-based production and purification of biologically active, hydrophobic and positively charged antimicrobial peptide.     

De novo designed synthetic mimics of antimicrobial peptides

Antimicrobial peptides are small cationic amphiphiles that play an important role in the innate immune system. Given their broad specificity, they appear to be ideal therapeutic agents. As a result, over the last decade, there has been considerable interest in developing them as intravenously administered antibiotics. However, it has proven difficult to accomplish this goal with peptide-based structures. Although it has been possible to solve some relatively simple problems such as susceptibility to proteolysis, more severe problems have included the expense of the materials, toxicity, limited efficacy, and limited tissue distribution. In an effort to overcome these problems, we developed small synthetic oligomers designed to adopt amphiphilic conformations and exhibit potent antimicrobial activity while being nontoxic to host cells. One class of these synthetic mimics of antimicrobial peptides (SMAMPs) is being developed as intravenous antibiotics.     

Designing the antimicrobial peptide with centrosymmetric and amphipathic characterizations for improving antimicrobial activity

Antibiotic-resistant bacterial infections are becoming a serious health issue and will cause 10 million deaths per year by 2050. As a result, the development of new antimicrobial agents is urgently needed. Antimicrobial peptides (AMPs) are found in the innate immune systems of various organisms to effectively fend off invading pathogens. In this study, we designed a series of AMPs (THL-2-1 to THL-2-9) with centrosymmetric and amphipathic properties, through substituting different amino acids on the hydrophobic side and at the centrosymmetric position to improve their antimicrobial activity. The results showed that leucine as a residue on the hydrophobic side of the peptide could enhance its antimicrobial activity and that glutamic acid as a centrosymmetric residue could increase the salt resistance of the peptide. Thus, the THL-2-3 peptide (KRLLRELKRLL-NH₂) showed the greatest antimicrobial activity (MIC₉₀ of 16 μM) against Gram-negative bacteria and had the highest salt resistance and cell selectivity among all the designed peptides. In summary, the results of this study provide useful references for the design of AMPs to enhance antimicrobial activity.     

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.     

Solution structure of nocistatin, a new peptide analgesic

Nocistatin, a new heptadecapeptide encoded in the bPNP-3 gene, has a powerful biological activity connected with the mechanisms of pain transmission. It does not bind to the opioid receptors but to another brain receptor with high affinity. In order to substantiate these novel biological data with a structural basis, we have undertaken a conformational study in solution. Proton nmr data in helicogenic solvents are consistent with a well-defined helical structure that is consistent with the nmr parameters of the C-terminal octapeptide, a shorter fragment that retains allodynia-blocking activity.     

Solution structure and membrane interaction mode of an antimicrobial peptide gaegurin 4

We have applied NMR spectroscopy to determine the high-resolution structure of gaegurin 4, a 37-residue antimicrobial peptide from Rana rugosa, under varying hydrophobic conditions. Even in 100% H2O, gaegurin 4 contains a nascent turn near its C-terminal Rana box. Under a more hydrophobic condition it forms two amphipathic helices, one long encompassing residues 2-23 and the other consisting of residues 25-34, similar to what has been observed in cecropin A. Functional implication of the helix-breaking kink at Gly24 in gaegurin 4 was investigated by preparing several analogs. Based upon the current and previous results, we propose a novel seaanemone-like ion pore-forming model for gaegurin 4.     

Solution structure of termite-derived antimicrobial peptide,spinigerin, as determined in SDS micelle by NMR spectroscopy

Spinigerin is a linear antibacterial peptide derived from a termite insect. It consists of 25 amino acids and is devoid of cysteines. Spinigerin displays good lytic activities against Gram-positive and Gram-negative bacteria, but has no hemolytic activities against human erythrocytes. In this study, we present a three-dimensional solution structure of spinigerin in SDS micelles. According to CD data spinigerin has an alpha-helical conformation in the presence of TFE, DPC micelles, and SDS micelles. The three-dimensional structure of spinigerin as determined by NMR spectroscopy contains a stable alpha-helix from Lys4 to Thr23. Spinigerin (4-21), an 18-residue fragment from Lys4 to Leu21, contains a similar content of alpha-helical structure compared to native spinigerin and was found to retain antibacterial activity, too. Therefore, this alpha-helical structure and the strong electrostatic attraction between four Lys and three Arg residues in spinigerin and the negatively charged polar head groups of the phospholipids on the membrane surface play important roles in disrupting membrane and subsequent cell death.     

Solution structure of microcin J25, the single macrocyclic antimicrobial peptide from Escherichia coli.

The three-dimensional solution structure of microcin J25, the single cyclic representative of the microcin antimicrobial peptide class produced by enteric bacteria, was determined using two-dimensional 1H NMR spectroscopy and molecular modeling. This hydrophobic 21-residue peptide exhibits potent activity directed to Gram-negative bacteria. Its primary structure, cyclo(-V1GIGTPISFY10GGGAGHVPEY20F-), has been determined previously [Blond, A., Péduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthélémy, M., Prigent, Y., Salomón, R.A., Farías, R.N., Moreno, F. & Rebuffat, S. (1999) Eur. J. Biochem., 259, 747-755]. Conformational parameters (3JNHCalphaH coupling constants, quantitative nuclear Overhauser enhancement data, chemical shift deviations, temperature coefficients of amide protons, NH-ND exchange rates) were obtained in methanol solution. Structural restraints consisting of 190 interproton distances inferred from NOE data, 11 phi backbone dihedral angle and 9 chi1 angle restraints derived from the coupling constants and three hydrogen bonds in agreement with the amide exchange rates were used as input for simulated annealing calculations and energy minimization in the program XPLOR. Microcin J25 adopts a well-defined compact structure consisting of a distorted antiparallel beta sheet, which is twisted and folded back on itself, thus resulting in three loops. Residues 7-10 and 17-20 form the more regular part of the beta sheet. The region encompassing residues Gly11-His16 consists of a distorted beta hairpin, which divides into two small loops and is stabilized by an inverse gamma turn and a type I' beta turn. The reversal of the chain leading to the Phe21-Pro6 loop results from a mixed beta/gamma turn. A cavity, in which the hydrophilic Ser8 side-chain is confined, is delimited by two crab pincer-like regions that comprise residues 6-8 and 18-1.     

Solution structure of antimicrobial peptide esculentin-1c from skin secretion of Rana esculenta

Granular glands in the skins of frogs synthesize and secrete a remarkably diverse range of peptides capable of antimicrobial activity. These anuran skin antimicrobial peptides are commonly hydrophobic, cationic and form an amphipathic α-helix in a membrane mimetic solution. Recently, they have been considered as useful target molecules for developing new antibiotics drugs. Esculentin-1c is a 46-amino acid residue peptide isolated from skin secretions of the European frog, Rana esculenta. It displays the most potent antimicrobial activity among bioactive molecules. Esculentin-1c has the longest amino acids among all antimicrobial peptides. The present study solved the solution structure of esculentin-1c in TFE/water by NMR, for the first time. We conclude that this peptide is comprised of three α-helices with each helix showing amphipathic characteristics, which seems to be a key part for permeating into bacterial membranes, thus presenting antimicrobial activity.     

Solution structure of humanin, a peptide against Alzheimer's disease-related neurotoxicity

Humanin is a newly identified 24-residue peptide that suppresses neuronal cell death caused by a wide spectrum of familial Alzheimer's disease genes and the beta-amyloid peptide. In this study, NMR and circular dichroism studies of synthetic humanin in aqueous and 30% 2,2,2-trifluoroethanol (TFE) solutions are reported. In aqueous solution, humanin exists predominantly in an unstructured conformation in equilibrium with turn-like structures involving residues Gly5 to Leu10 and Glu15 to Leu18, providing indication of nascent helix. In the less polar environment of 30% TFE, humanin readily adopts helical structure with long-range order spanning residues Gly5 to Leu18. Comparative 3D modeling studies and topology predictions are in qualitative agreement with the experimental findings in both environments. Our studies reveal a flexible peptide in aqueous environment, which is free to interact with possible receptors that mediate its action, but may also acquire a helical conformation necessary for specific interactions and/or passage through membranes.     

A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis

Several synthetic class A peptide analogues have been shown to mimic many of the properties of human apo A-I in vitro. A new peptide [acetyl-(AspTrpLeuLysAlaPheTyrAspLysValPheGluLysPheLysGluPhePhe)-NH2; 5F], with increased amphipathicity, was administered by intraperitoneal injection, 20 microg/day for 16 weeks, to C57BL/6J mice fed an atherogenic diet. Mouse apo A-I (MoA-I) (50 microg/day) or phosphate-buffered saline (PBS) injections were given to other mice as controls. Total plasma cholesterol levels and lipoprotein profiles were not significantly different between the treated and control groups, except that the mice receiving 5F or MoA-I had lower high density lipoprotein (HDL) cholesterol when calculated as a percentage of total cholesterol. No toxicity or production of antibodies to the injected materials was observed. When HDL was isolated from high fat diet-administered mice injected with 5F and presented to human artery wall cells in vitro together with human low density lipoprotein (LDL), there were substantially fewer lipid hydroperoxides formed and substantially less LDL-induced monocyte chemotactic activity than with HDL from PBS-injected animals. Injection of human apo A-I produced effects similar to 5F on lipid peroxide formation and LDL-induced monocyte chemotactic activity, but injection of MoA-I was significantly less effective in reducing lipid hydroperoxide formation or lowering LDL-induced monocyte chemotactic activity. Mice receiving peptide 5F had significantly less aortic atherosclerotic lesion area compared with mice receiving PBS, whereas lesion area in mice receiving MoA-I was similar to that of the PBS-injected animals. This is the first in vivo demonstration that a model class A amphipathic helical peptide has antiatherosclerotic properties. We conclude that 5F inhibits lesion formation in high fat diet-administered mice by a mechanism that does not involve changes in the lipoprotein profile, and may have potential in the prevention and treatment of atherosclerosis.