Structure, activity and interactions of the cysteine deleted analog of tachyplesin-1 with lipopolysaccharide micelle: Mechanistic insights into outer-membrane permeabilization and endotoxin neutralization

AbstractTachyplesin-1, a disulfide stabilized β-hairpin antimicrobial peptide, can be found at the hemocytes of horse shoe crab Tachypleus tridentatus. A cysteine deleted linear analog of tachyplesin-1 or CDT (KWFRVYRGIYRRR-NH2) contains a broad spectrum of bactericidal activity with a reduced hemolytic property. The bactericidal activity of CDT stems from selective interactions with the negatively charged lipids including LPS. In this work, CDT–LPS interactions were investigated using NMR spectroscopy, optical spectroscopy and functional assays. We found that CDT neutralized LPS and disrupted permeability barrier of the outer membrane. Zeta potential and ITC studies demonstrated charge compensation and hydrophobic interactions of CDT with the LPS-outer membrane, respectively. Secondary structure of the peptide was probed by CD and FT-IR experiments indicating β-strands and/or β-turn conformations in the LPS micelle. An ensemble of structures, determined in LPS micelle by NMR, revealed a β-hairpin like topology of the CDT peptide that was typified by an extended cationic surface and a relatively shorter segment of hydrophobic region. Interestingly, at the non-polar face, residue R11 was found to be in a close proximity to the indole ring of W2, suggesting a cation–π type interactions. Further, saturation transfer difference (STD) NMR studies established intimate contacts among the aromatic and cationic residues of CDT with the LPS micelle. Fluorescence and dynamic light scattering experiments demonstrated that CDT imparted structural destabilization to the aggregated states of LPS. Collectively, atomic resolution structure and interactions of CDT with the outer membrane-LPS could be exploited for developing potent broad spectrum antimicrobial and anti-sepsis agents

β-Boomerang Antimicrobial and Antiendotoxic Peptides: Lipidation and Disulfide Bond Effects on Activity and Structure

Drug-resistant Gram-negative bacterial pathogens and endotoxin- or lipopolysaccharide (LPS)-mediated inflammations are among some of the most prominent health issues globally. Antimicrobial peptides (AMPs) are eminent molecules that can kill drug-resistant strains and neutralize LPS toxicity. LPS, the outer layer of the outer membrane of Gram-negative bacteria safeguards cell integrity against hydrophobic compounds, including antibiotics and AMPs. Apart from maintaining structural integrity, LPS, when released into the blood stream, also induces inflammatory pathways leading to septic shock. In previous works, we have reported the de novo design of a set of 12-amino acid long cationic/hydrophobic peptides for LPS binding and activity. These peptides adopt β-boomerang like conformations in complex with LPS. Structure-activity studies demonstrated some critical features of the β-boomerang scaffold that may be utilized for the further development of potent analogs. In this work, β-boomerang lipopeptides were designed and structure-activity correlation studies were carried out. These lipopeptides were homo-dimerized through a disulfide bridge to stabilize conformations and for improved activity. The designed peptides exhibited potent antibacterial activity and efficiently neutralized LPS toxicity under in vitro assays. NMR structure of C4YI13C in aqueous solution demonstrated the conserved folding of the lipopeptide with a boomerang aromatic lock stabilized with disulfide bond at the C-terminus and acylation at the N-terminus. These lipo-peptides displaying bacterial sterilization and low hemolytic activity may be useful for future applications as antimicrobial and antiendotoxin molecules

Designed antimicrobial peptides : structure-activity correlations and mode of action

The discovery of antibiotics and its incredible usage in saving human lives has been considered as one of the major discoveries in medicine. However, fight against bacteria has not ended due to the emergence of resistant microbes. Super bugs or Multi- drug resistance (MDR) pathogens have become a common occurrence now. The situation is further complicated by the outburst of new diseases like H1N1, Dengue etc and its associated illnesses. The immune suppression of host system during such illness also invites other hospital infections and hence the treatment involves usage of multiple drugs. The mode of action of antibiotics is receptor mediated and hence there are high chances for the microbes to mutate such receptors and become resistance to that drug. Under such circumstances, the discovery of antimicrobial peptides incited a new path in development of future drugs, which the microbes would find it difficult to develop resistance. Lipopolysaccharides (LPS) are present in the outer layer of outer membrane of Gram negative bacteria. It serves dual role, as an important component in maintaining structural integrity of bacteria and also as membrane impermeability barrier to antibiotics and other hydrophobic substances. LPS is released in to the blood stream either during cell division or by the action of antibiotics. Such circulating LPS when accumulated in higher amounts were recognized to stimulate inflammatory pathway provoking septic shock syndrome. Sepsis is identified as one of the major mortality causing condition in hospital Intensive care units (ICU). Activity studies of antimicrobial peptides (AMPs) revealed that they could bind and neutralize the LPS before exerting its killing activity. Hence these AMPs from natural sources can be used as template for the synthesis of effective antiendotoxic antimicrobial peptides. In the current work, antimicrobial peptides were designed to characterize the structural features that govern the endotoxin neutralization and bacterial killing. First and second generation-boomerang peptides showed promising antimicrobial activity with LPS neutralization. Since N-terminal acylation and dimerization was found to increase antimicrobial activity. -boomerang peptides were acylated and dimerized through disulfide bridges in third generation. Isoleucine analogs (devoid of cysteine mediated disulphide link, YI13WF) and Cysteine analogs (YI13WFC) were further characterized for antiendotoxic antimicrobial activity. Both the analogs were also acylated with different length of acyl chains to define the appropriate hydrophobicity limit for the activity of the lipopeptides thus designed. Structure- activity correlation studies of the peptides explained that the cysteine analogs exhibited more efficient anti microbial activity than the isoleucine analogs. Further, the dimerized lipopeptides (acylated cysteine analogs) demonstrated broad spectrum of antibacterial activity with efficient LPS perturbation. Among the different chain lengths for acylation, C4 was found to be more appropriate as the increase in hydrophobicity complemented with antimicrobial antiendotoxic activity with reduced hemolytic activity. Atomic resolution structure of C4YI13WFC in aqueous state revealed that the peptide folds into characteristic boomerang shape with disulfide linkage between two cysteine residue bringing the residues W4 and F12 close together to form the aromatic fold. In the second project, the structured LPS binding motif (GG8WF) identified in second generation of -boomerang peptides was conjugated with the peptides that are found to aggregate on LPS layer, but active once trespasses the LPS layer. GG8WF peptide also found to fold into characteristic boomerang shape with aromatic fold between W2 and F7 upon interaction with LPS. GG8WF peptide was conjugated at C-terminus of Temporin A, Temporin B and synthetic K5L7 peptides that are found to aggregate on LPS layer. Three dimensional structure determination of the hybrid peptide revealed that the peptide folds into alpha helix upon membrane interaction and the positive charges in GG8 peptide initiates the binding of peptide with outer membrane. It was also found that the conjugation rendered alpha helix to boomerang motif also, but instead of the conserved aromatic lock between W2 and F7, cation-π interaction between W15 and R19 (in hybrid peptides) accounted for the remarkable activity of the hybrid peptides. When this R19 was mutated to alanine, it produced an inactive peptide. In the third project, cysteine deleted analogs of Protegrin-1 peptide, isolated from porcine leukocytes were characterized. The full length analog without cysteine, RR14 was found to be active followed by truncated analogs RR11 and LR10. The beta hairpin fold with π- π interactions between Tyr and Phe was found to be conserved, even after deletion of cysteine residue. SAR studies of these analogs revealed that the beta hairpin fold is important in exerting the antibacterial activity and that the first four residues RGGR provides the initial ionic interaction with outer membrane. When RGGR was deleted, the peptide showed impaired antibacterial activity. In the last project, antimicrobial peptides rich in arginine with appropriately placed tryptophan, leucine and isoleucine residues were designed. Increase in hydrophobic surface of the peptide confers salt resistance to antimicrobial peptides. Pharmacological development of antimicrobial peptides requires the peptide to be salt tolerant. These series of designed peptides were found to be salt tolerant and adopt alpha helix upon interaction with LPS outer membrane.BIOLOGICAL SCIENCE

Resurrecting inactive antimicrobial peptides from the lipopolysaccharide trap

Host defense antimicrobial peptides (AMPs) are a promising source of antibiotics for the treatment of multiple-drug-resistant pathogens. Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, functions as a permeability barrier against a variety of molecules, including AMPs. Further, LPS or endotoxin is the causative agent of sepsis killing 100,000 people per year in the United States alone. LPS can restrict the activity of AMPs inducing aggregations at the outer membrane, as observed for frog AMPs, temporins, and also in model AMPs. Aggregated AMPs, "trapped" by the outer membrane, are unable to traverse the cell wall, causing their inactivation. In this work, we show that these inactive AMPs can overcome LPS-induced aggregations while conjugated with a short LPS binding β-boomerang peptide motif and become highly bactericidal. The generated hybrid peptides exhibit activity against Gram-negative and Gram-positive bacteria in high-salt conditions and detoxify endotoxin. Structural and biophysical studies establish the mechanism of action of these peptides in LPS outer membrane. Most importantly, this study provides a new concept for the development of a potent broad-spectrum antibiotic with efficient outer membrane disruption as the mode of action.Accepted versio

Cysteine deleted protegrin-1 (CDP-1) : anti-bacterial activity, outer-membrane disruption and selectivity

BACKGROUND: Protegin-1 (PG-1: RGGRLCYCRRRFCVCVGR-amide) assumes a rigid β-hairpin like structure that is stabilized by two disulfide bridges between Cys6-Cys15 and Cys8-Cys13. Previous studies, employing linear analogs of PG-1, with Cys to Ala mutations or modified Cys, have demonstrated that the disulfide bridges are critical for the broad spectrum and salt resistant antimicrobial activity of PG-1. METHODS: In order to understand structural and functional roles of disulfide bonds in protegrins, we have synthesized a Cys deleted variant of PG-1 or CDP-1, RGGRLYRRRFVVGR-amide, and two of its analogs, RR11, RLYRRRFVVGR-amide, and LR10, LYRRRFVVGR-amide, containing deletion of residues at the N-terminus. These peptides have been characterized for bactericidal activity and mode of action in lipopolysaccharide (LPS) using optical spectroscopy, ITC and NMR. RESULTS: Antibacterial activity, against Gram-negative and Gram-positive strains, of the three peptides follows the order: CDP-1>RR11>LR10. LR10 displays only limited activity toward Gram-negative strains. CDP-1 demonstrates efficient membrane permeabilization and high-affinity interactions with LPS. CDP-1 and RR11 both assume β-hairpin like compact structures in complex with LPS, whereas LR10 adopts an extended conformation in LPS. In zwitterionic DPC micelles CDP-1 and the truncated analog peptides do not adopt folded conformations. MAJOR CONCLUSIONS: Despite the absence of stabilizing disulfide bridges CDP-1 shows broad-spectrum antibacterial activity and assumes β-hairpin like structure in complex with LPS. The β-hairpin structure may be essential for outer membrane permeabilization and cell killing.Accepted versio

Fast and Green Synthesis of an Oligo-Hydrocaffeic Acid-Based Adhesive

A green, mussel-inspired bioadhesive based on oligomerization of hydrocaffeic acid was synthesized in water by an ultrafast one-step reaction in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as an activating agent. The resulting oligomers exhibited strong wet adhesion when applied to different substrates including glass, stainless steel, and aluminum. Compared to most commercial adhesives, this bioinspired adhesive is produced via a sustainable and green process, i.e., aqueous-based synthesis, one-step reaction, and in the absence of any purification step to obtain the final functional adhesive.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

Self-assembly of a barnacle cement protein (MrCP20) into adhesive nanofibrils with concomitant regulation of CaCO₃ polymorphism

Barnacles are convenient model organisms to study both biomineralization and bioadhesion phenomena. They secrete a proteinaceous adhesive from cement proteins (CPs) to strongly attach to solid immersed substrates. More recently, it has been suggested that some CPs also play a key role in regulating the calcification of barnacles’ protective shells. In this study, combining both solution and solid-state NMR spectroscopy, Raman and infrared spectroscopy studies, and atomic force microscopy (AFM) and transmission electron microscopy (TEM) imaging, we have explored the CaCO3 mineralization pathway regulated by Megabalanus rosa CP 20 (MrCP20). Our data show that MrCP20 can sequestrate inorganic calcium and carbonate ions from the solution state, which quickly coarsen into liquid-like microdroplets and subsequently form protovaterite amorphous CaCO3 (ACC) particles. This pathway leads to the stabilization of the metastable vaterite polymorphism of CaCO3. Simultaneously, AFM and TEM investigations show that MrCP20 undergoes fibrillization triggered by a pH drift arising during CaCO3 mineralization, leading to amyloid-like nanofibrils. Based on protein NMR, this mechanism appears to be stabilized by the reduction of intramolecular disulfide bonds. Collectively, our results demonstrate that MrCP20 plays a synergistic role of regulating CaCO3 biomineralization while concomitantly self-assembling into adhesive nanofibrils.Ministry of Education (MOE)H.M. and A.M. acknowledge funding from the US Office of Naval Research-Global (ONR-G), grant no. N62909-17-1- 2045, and the Singapore Ministry of Education (MOE) through an Academic Research Fund (AcRF) tier 3 grant (# MOE 2019-T3-1-012). T.G. and T.A. acknowledge funding from CNRS through the NTU-CNRS “Excellence Science” joint research program (no. 294 382)

NMR structure, localization, and vesicle fusion of Chikungunya virus fusion peptide

The virus–host cell fusion process is mediated by a membrane anchored viral fusion protein that inserts its hydrophobic fusion peptide into the plasma membrane of the host cell, initiating the fusion reaction. Therefore, fusion peptides are an important functional constituent of the fusion proteins of enveloped viruses. In this work, we characterize the fusion peptide or VT18 (V84YPFMWGGAYCFCDAENT101) of Chikungunya virus (CHIKV) using NMR and fluorescence spectroscopy in zwitterionic lipid environments. Our results demonstrate that the VT18 peptide is able to induce liposome fusions in a pH independent manner and interacts with the zwitterionic lipid vesicles. The NMR derived three-dimensional structure of VT18, in solution of dodecylphosphocholine (DPC) micelles, is typified by extended or β-type conformations for most of the residues, whereby residues M88-W89-G90-G91 adopt a type I β-turn conformation. Strikingly, the aromatic side chains of residues Y85, F87, Y93, and F95 in the VT18 structure are found to be well-packed forming an aromatic core. In particular, residue F87 is situated at the center of the aromatic core establishing a close proximity with other aromatic side chains. Further, the aromatic core residues are also involved in packing interactions with the side chains of residues M88, C94. Paramagnetic relaxation enhancement NMR, using spin labeled doxyl lipids, indicated that the aromatic core residues of VT18 are well inserted into the micelles, whereas the polar residues at the C-terminus may be surface localized. The atomic resolution structure and lipid interactions of CHIKV fusion peptide presented here will aid to uncover the fusion mechanism by the type II viral fusion proteins

Three-dimensional structure of Megabalanus rosa cement protein 20 revealed by multi-dimensional NMR and molecular dynamics simulations

Barnacles employ a protein-based cement to firmly attach to immersed substrates. The cement proteins (CPs) have previously been identified and sequenced. However, the molecular mechanisms of adhesion are not well understood, in particular, because the three-dimensional molecular structure of CPs remained unknown to date. Here, we conducted multi-dimensional nuclear magnetic resonance (NMR) studies and molecular dynamics (MD) simulations of recombinant Megabalanus rosa Cement Protein 20 (rMrCP20). Our NMR results show that rMrCP20 contains three main folded domain regions intervened by two dynamic loops, resulting in multiple protein conformations that exist in equilibrium. We found that 12 out of 32 Cys in the sequence engage in disulfide bonds that stabilize the β-sheet domains owing to their placement at the extremities of β-strands. Another feature unveiled by NMR is the location of basic residues in turn regions that are exposed to the solvent, playing an important role for intermolecular contact with negatively charged surfaces. MD simulations highlight a highly stable and conserved β-motif (β7-β8), which may function as nuclei for amyloid-like nanofibrils previously observed in the cured adhesive cement. To the best of our knowledge, this is the first report describing the tertiary structure of an extracellular biological adhesive protein at the molecular level. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.This study was funded by the US Office of Naval Research – Global (ONR-G), grant no. N62909-17-1-2045

NMR Structure of Temporin-1 Ta in Lipopolysaccharide Micelles: Mechanistic Insight into Inactivation by Outer Membrane

Antimicrobial peptides (AMPs) play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS). The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane.Using NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide) in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD) NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles.We propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a broader spectrum of activity