Harini Mohanram

Designed β-Boomerang Antiendotoxic and Antimicrobial Peptides: STRUCTURES AND ACTIVITIES IN LIPOPOLYSACCHARIDE*♦

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like beta-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nM concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the beta-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate beta-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nm concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.

NMR Structures and Interactions of Temporin-1Tl and Temporin-1Tb with Lipopolysaccharide Micelles MECHANISTIC INSIGHTS INTO OUTER MEMBRANE PERMEABILIZATION AND SYNERGISTIC ACTIVITY

Temporins are a group of closely related short antimicrobial peptides from frog skin. Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, plays important roles in the activity of temporins. Earlier studies have found that LPS induces oligomerization of temporin-1Tb (TB) thus preventing its translocation across the outer membrane and, as a result, reduces its activity on Gram-negative bacteria. On the other hand, temporin-1Tl (TL) exhibits higher activity, presumably because of lack of such oligomerization. A synergistic mechanism was proposed, involving TL and TB in overcoming the LPS-mediated barrier. Here, to gain insights into interactions of TL and TB within LPS, we investigated the structures and interactions of TL, TB, and TL+TB in LPS micelles, using NMR and fluorescence spectroscopy. In the context of LPS, TL assumes a novel antiparallel dimeric helical structure sustained by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. By contrast, independent TB has populations of helical and aggregated conformations in LPS. The LPS-induced aggregated states of TB are largely destabilized in the presence of TL. Saturation transfer difference NMR studies have delineated residues of TL and TB in close contact with LPS and enhanced interactions of these two peptides with LPS, when combined together. Fluorescence resonance energy transfer and 31P NMR have pointed out the proximity of TL and TB in LPS and conformational changes of LPS, respectively. Importantly, these results provide the first structural insights into the mode of action and synergism of antimicrobial peptides at the level of the LPS-outer membrane.

Lipopolysaccharide bound structures of the active fragments of fowlicidin‐1, a cathelicidin family of antimicrobial and antiendotoxic peptide from chicken, determined by transferred nuclear overhauser effect spectroscopy

Cathelicidins comprise a major family of host‐defense antimicrobial peptides in vertebrates. The C‐terminal part of the cathelicidins is bestowed with antimicrobial and lipopolysaccharide (LPS) neutralizing activities. In this work, we repot high resolution solution structures of two nontoxic active fragments, residues 1–16 or RG16 and residues 8–26 or LK19, of fowlicidin‐1, a cathelicidin family of peptide from chicken, as a complex with LPS using two‐dimensional transferred nuclear Overhauser effect (Tr‐NOE) spectroscopy. Both peptides are highly flexible and do not assume any preferred conformations in their free states. Upon complexation with endotoxin or LPS, peptides undergo structural transitions towards folded conformations. Structure calculations reveal that the LK19 peptide adopts a well defined helical structure with a bend at the middle. By contrast, the first seven amino acids of RG16 are found to be flexible followed by a helical conformation for the residues L8‐A15. In addition, a truncated version of LK19 encompassing residues A15‐K26 or AK12 displays an amphipathic helical structure in LPS. Saturation transfer difference (STD) NMR studies demonstrate that all peptides, RG16, LK19, and AK12, are in close proximity with LPS, whereby the aromatic residues showed the strongest STD effects. Fluorescence studies with fluorescein isothiocyanate (FITC) labeled LPS in the presence of full‐length fowlicidin‐1, LK19, RG16, and AK12 indicated that LPS‐neutralization property of these peptides may result from plausible dissociation of LPS aggregates. The helical structures of peptide fragments derived from fowlicidin‐1 in LPS could be utilized to develop nontoxic antiendotoxic compounds.

Design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt‐resistance, and biocompatibility

Antimicrobial peptides (AMPs) kill microbes by non‐specific membrane permeabilization, making them ideal templates for designing novel peptide‐based antibiotics that can combat multi‐drug resistant pathogens. For maximum efficacy in vivo and in vitro, AMPs must be biocompatible, salt‐tolerant and possess broad‐spectrum antimicrobial activity. These attributes can be obtained by rational design of peptides guided by good understanding of peptide structure‐function. Toward this end, this study investigates the influence of charge and hydrophobicity on the activity of tryptophan and arginine rich decamer peptides engineered from a salt resistant human β‐defensin‐28 variant. Mechanistic investigations of the decamers with detergents mimicking the composition of bacterial and mammalian membrane, reveal a correlation between improved antibacterial activity and the increase in tryptophan and positive residue content, while keeping hemolysis low. The potent antimicrobial activity and high cell membrane selective behavior of the two most active decamers, D5 and D6, are attributed to an optimum peptide charge to hydrophobic ratio bestowed by systematic arginine and tryptophan substitution. D5 and D6 show surface localization behavior with binding constants of 1.86 × 108 and 2.6 × 108 M−1, respectively, as determined by isothermal calorimetry measurements. NMR derived structures of D5 and D6 in SDS detergent micelles revealed proximity of Trp and Arg residues in an extended structural scaffold. Such potential cation–π interactions may be critical in cell permeabilization of the AMPs. The fundamental characterization of the engineered decamers provided in this study improves the understanding of structure–activity relationship of short arginine tryptophan rich AMPs, which will pave the way for future de novo design of potent AMPs for therapeutic and biomedical applications. Biotechnol. Bioeng. 2014;111: 37–49.

Resurrecting Inactive Antimicrobial Peptides from the Lipopolysaccharide Trap

ABSTRACT 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.

NMR Solution Conformations and Interactions of Integrin αLβ2 Cytoplasmic Tails

The integrins are bi-directional signal transducers. Devoid of enzymatic activity, the integrin cytoplasmic tail serves as a hub for the recruitment of cytosolic proteins, and many of these are signaling molecules. The leukocyte-restricted integrin αLβ2 is essential for the adhesion, migration, and proliferation of leukocytes. Here we report solution conformations and interactions of the αLβ2 cytoplasmic tails by NMR analyses. The αL tail is characterized by three helical segments in the order of helix 1-3 that are connected by two loops with helix 3 having a number of nuclear Overhauser effect contacts with helix 1 and helix 2. The conformation of the β2 tail is less defined with only a helical segment restricted at its N terminus. Acidic residues from the helix 2-loop-helix 3 motif of αL were found to be responsible for its binding to calcium ion. There were detectable interactions between αL and β2 tails, involving helix 1 and helix 3 of the αL tail and the N-terminal helix of the β2 tail. Talin head domain that contains the FERM domain showed binding affinity of Kd ∼ 0.5 μm with the β2 tail. The binding affinity of αL and β2 tails is Kd ∼ 2.63 μm. These data are in line with the activating property of talin head domain on αLβ2 by which binding of talin head domain to β2 tail disrupts the interface of the αL and β2 tails that constrains αLβ2 in a resting state.

NMR structural studies of the Ste11 SAM domain in the dodecyl phosphocholine micelle.

The sterile α‐motif (SAM), a relatively small (∼70 amino acids) interaction domain, is found in a variety of proteins involved in cell signaling, transcription regulation, and scaffolding. The Ste11 protein kinase from the mitogen activated protein kinase (MAPK) signaling cascades of the budding yeast is regulated by a SAM domain located at the N‐terminus of full‐length protein. In solution, the Ste11 SAM domain exists as a well‐folded dimeric structure that is involved in interaction with the cognate SAM domain from an adaptor protein Ste50. In this work, we show that the Ste11 SAM domain has an intrinsic affinity towards the lipid membranes. The solution conformation of the Ste11 SAM determined in perdeuterated DPC micelle, using NMR spectroscopy, is defined by five helices of different lengths connected by a number of loops. In the micelle bound state, the non‐polar and aromatic residues of the Ste11 SAM lack a native‐like packing and are presumably engaged in interactions with the micelle. Using two different paramagnetic doxyl‐lipids; we have mapped out localization of Ste11 SAM residues at the micelle surface. Most of the residues appear to localize at the interfacial region of the micelle. However, a number of non‐polar residues from the central region of the domain are found to be located inside the core of the micelle including residues from the helix 4 and a loop between helix 2 and helix 3. Isothermal titration calorimetry studies demonstrate that a facile insertion of the Ste11 SAM into the DPC micelle is primarily driven by a large change in enthalpy, −50 kcal/mol with an apparent equilibrium association constant (Ka) of 7.86 × 106 M−1. Interestingly, an interfacial mutant L60R of the Ste11 SAM lacking the dimeric structure does not show detectable interactions with the lipid micelle. The micelle‐bound structure of the Ste11 SAM domain described in this work may have potential implications in the regulation of MAPK signaling whereby positioning of the Ste11 protein in close proximity to the membrane may facilitate efficient phosphorylation of the Ste11 kinase by the membrane attached upstream Ste20/pak kinase. Proteins 2009.

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

Background 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. Principal Findings 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. Significance 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.

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.

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.