Outer-Membrane Permeabilization, LPS Transport Inhibition: Activity, Interactions, and Structures of Thanatin Derived Antimicrobial Peptides.

Currently, viable antibiotics available to mitigate infections caused by drug-resistant Gram-negative bacteria are highly limited. Thanatin, a 21-residue-long insect-derived antimicrobial peptide (AMP), is a promising lead molecule for the potential development of novel antibiotics. Thanatin is extremely potent, particularly against the Enterobacter group of Gram-negative pathogens, e.g., E. coli and K. pneumoniae. As a mode of action, cationic thanatin efficiently permeabilizes the LPS-outer membrane and binds to the periplasmic protein LptAm to inhibit outer membrane biogenesis. Here, we have utilized N-terminal truncated 16- and 14-residue peptide fragments of thanatin and investigated structure, activity, and selectivity with correlating modes of action. A designed 16-residue peptide containing D-Lys (dk) named VF16 (V1PIIYCNRRT-dk-KCQRF16) demonstrated killing activity in Gram-negative bacteria. The VF16 peptide did not show any detectable toxicity to the HEK 293T cell line and kidney cell line Hep G2. As a mode of action, VF16 interacted with LPS, permeabilizing the outer membrane and binding to LptAm with high affinity. Atomic-resolution structures of VF16 in complex with LPS revealed cationic and aromatic surfaces involved in outer membrane interactions and permeabilization. Further, analyses of an inactive 14-residue native thanatin peptide (IM14: IIYCNRRTGKCQRM) delineated the requirement of the ß-sheet structure in activity and target interactions. Taken together, this work would pave the way for the designing of short analogs of thanatin-based antimicrobials.

Membrane cholesterol regulates the oligomerization and fusogenicity of SARS-CoV fusion peptide: implications in viral entry.

N-terminal residues (770-788) of the S2 glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) have been recognized as a potential fusion peptide that can be involved in the entry of the virus into the host cell. Membrane composition plays an important role in lipid-peptide interaction and the oligomeric status of the peptide. SARS-CoV fusion peptide (S2 fusion peptide) is known to undergo cholesterol-dependent oligomerization in the membrane; however, its significance in membrane fusion is still speculative. This study aimed to investigate the oligomerization of SARS-CoV fusion peptide in a membrane containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with varying concentrations of cholesterol, and to evaluate peptide-induced membrane fusion to correlate the importance of peptide oligomerization with membrane fusion. Peptide-induced modulation of membrane organization and dynamics was explored by steady-state and time-resolved fluorescence spectroscopic measurements using depth-dependent probes. The results clearly demonstrated the induction of S2 fusion peptide oligomerization by membrane cholesterol and the higher efficiency of the oligomer in promoting membrane fusion compared to its monomeric counterpart. Cholesterol-dependent peptide oligomerization and membrane fusion are important aspects of viral infection since the cholesterol level can change with age as well as with the onset of various pathophysiological conditions.

NMR Structure and Localization of the Host Defense Peptide ThanatinM21F in Zwitterionic Dodecylphosphocholine Micelle: Implications in Antimicrobial and Hemolytic Activity.

Non-hemolytic antimicrobial peptides (AMPs) are vital lead molecules for the designing and development of peptide-based antibiotics. Thanatin a 21-amino acid long single disulfide bonded AMP is known to be highly non-hemolytic with a limited toxicity to human cells and model animals. Thanatin demonstrates a potent antibacterial activity against multidrug-resistant Gram-negative pathogens. A single mutated variant of thanatin replaced last residue Met21 to Phe or thanatin M21F has recently been found to be more active compared to the native peptide. In order to gain mechanistic insights toward bacterial cell lysis versus non-hemolysis, here, we report atomic resolution structure and mode insertion of thanatinM21F reconstituted into zwitterionic detergent micelle by use of solution NMR spectroscopy. The 3D structure of thanatinM21F in DPC micelle is defined by an anti-parallel ß-sheet between residues I9-F21 with a central cationic loop, residues N12-R14. PRE NMR studies revealed hydrophobic core residues of thanatinM21F are deeply inserted in the DPC micelle, while residues at the extended N-terminal half of the peptide are appeared to be mostly surface localized. Marked structural differences of thanatin and thanatinM21F in negatively charged LPS and DPC micelle could be correlated with non-hemolytic and antibacterial activity.

Atomic-Resolution Structures and Mode of Action of Clinically Relevant Antimicrobial Peptides.

Global rise of infections and deaths caused by drug-resistant bacterial pathogens are among the unmet medical needs. In an age of drying pipeline of novel antibiotics to treat bacterial infections, antimicrobial peptides (AMPs) are proven to be valid therapeutics modalities. Direct in vivo applications of many AMPs could be challenging; however, works are demonstrating encouraging results for some of them. In this review article, we discussed 3-D structures of potent AMPs e.g., polymyxin, thanatin, MSI, protegrin, OMPTA in complex with bacterial targets and their mode of actions. Studies on human peptide LL37 and de novo-designed peptides are also discussed. We have focused on AMPs which are effective against drug-resistant Gram-negative bacteria. Since treatment options for the infections caused by super bugs of Gram-negative bacteria are now extremely limited. We also summarize some of the pertinent challenges in the field of clinical trials of AMPs.

Ultra-Short Cyclized ß-Boomerang Peptides: Structures, Interactions with Lipopolysaccharide, Antibiotic Potentiator and Wound Healing.

Many antibiotics are ineffective in killing Gram-negative bacteria due to the permeability barrier of the outer-membrane LPS. Infections caused by multi-drug-resistant Gram-negative pathogens require new antibiotics, which are often difficult to develop. Antibiotic potentiators disrupt outer-membrane LPS and can assist the entry of large-scaffold antibiotics to the bacterial targets. In this work, we designed a backbone-cyclized ultra-short, six-amino-acid-long (WKRKRY) peptide, termed cWY6 from LPS binding motif of ß-boomerang bactericidal peptides. The cWY6 peptide does not exhibit any antimicrobial activity; however, it is able to permeabilize the LPS outer membrane. Our results demonstrate the antibiotic potentiator activity in the designed cWY6 peptide for several conventional antibiotics (vancomycin, rifampicin, erythromycin, novobiocin and azithromycin). Remarkably, the short cWY6 peptide exhibits wound-healing activity in in vitro assays. NMR, computational docking and biophysical studies describe the atomic-resolution structure of the peptide in complex with LPS and mode of action in disrupting the outer membrane. The dual activities of cWY6 peptide hold high promise for further translation to therapeutics.

De Novo-Designed ß-Sheet Heme Proteins.

The field of de novo protein design has met with considerable success over the past few decades. Heme, a cofactor, has often been introduced to impart a diverse array of functions to a protein, ranging from electron transport to respiration. In nature, heme is found to occur predominantly in α-helical structures over ß-sheets, which has resulted in significant designs of heme proteins utilizing coiled-coil helices. By contrast, there are only a few known ß-sheet proteins that bind heme and designs of ß-sheets frequently result in amyloid-like aggregates. This review reflects on our success in designing a series of multistranded ß-sheet heme binding peptides that are well folded in both aqueous and membrane-like environments. Initially, we designed a ß-hairpin peptide that self-assembles to bind heme and performs peroxidase activity in membrane. The ß-hairpin was optimized further to accommodate a heme binding pocket within multistranded ß-sheets for catalysis and electron transfer in membranes. Furthermore, we de novo designed and characterized ß-sheet peptides and miniproteins that are soluble in an aqueous environment capable of binding single and multiple hemes with high affinity and stability. Collectively, these studies highlight the substantial progress made toward the design of functional ß-sheets.

Enhanced Cholesterol-Dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to Its N-Terminal Counterpart.

Membrane fusion is an important step for the entry of the lipid-sheathed viruses into the host cells. The fusion process is being carried out by fusion proteins present in the viral envelope. The class I virus contains a 20-25 amino acid sequence at its N-terminal of the fusion domain, which is instrumental in fusion and is called as a "fusion peptide". However, severe acute respiratory syndrome (SARS) coronaviruses contain more than one fusion peptide sequences. We have shown that the internal fusion peptide 1 (IFP1) of SARS-CoV-2 is far more efficient than its N-terminal counterpart (FP) to induce hemifusion between small unilamellar vesicles. Moreover, the ability of IFP1 to induce hemifusion formation increases dramatically with growing cholesterol content in the membrane. Interestingly, IFP1 is capable of inducing hemifusion but fails to open the pore.

Thanatin: An Emerging Host Defense Antimicrobial Peptide with Multiple Modes of Action.

Antimicrobial peptides (AMPs) possess great potential for combating drug-resistant bacteria. Thanatin is a pathogen-inducible single-disulfide-bond-containing ß-hairpin AMP which was first isolated from the insect Podisus maculiventris. The 21-residue-long thanatin displays broad-spectrum activity against both Gram-negative and Gram-positive bacteria as well as against various species of fungi. Remarkably, thanatin was found to be highly potent in inhibiting the growth of bacteria and fungi at considerably low concentrations. Although thanatin was isolated around 25 years ago, only recently has there been a pronounced interest in understanding its mode of action and activity against drug-resistant bacteria. In this review, multiple modes of action of thanatin in killing bacteria and in vivo activity, therapeutic potential are discussed. This promising AMP requires further research for the development of novel molecules for the treatment of infections caused by drug resistant pathogens.

Nγ -Hydroxyasparagine: A Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases.

Peptidyl asparaginyl ligases (PALs) are powerful tools for peptide macrocyclization. Herein, we report that a derivative of Asn, namely Nγ -hydroxyasparagine or Asn(OH), is an unnatural P1 substrate of PALs. By Asn(OH)-mediated cyclization, we prepared cyclic peptides as new matrix metalloproteinase 2 (MMP2) inhibitors displaying the hydroxamic acid moiety of Asn(OH) as the key pharmacophore. The most potent cyclic peptide (Ki =2.8±0.5 nM) was built on the hyperstable tetracyclic scaffold of rhesus theta defensin-1. The Asn(OH) residue in the cyclized peptides can also be readily oxidized to Asp. By this approach, we synthesized several bioactive Asp-containing cyclic peptides (MCoTI-II, kB2, SFTI, and integrin-targeting RGD peptides) that are otherwise difficult targets for PAL-catalyzed cyclization owing to unfavorable kinetics of the P1-Asp substrates. This study demonstrates that substrate engineering is a useful strategy to expand the application of PAL ligation in the synthesis of therapeutic cyclic peptides.

Salt Dependence Conformational Stability of the Dimeric SAM Domain of MAPKKK Ste11 from Budding Yeast: A Native-State H/D Exchange NMR Study.

The sterile α motif, also called the SAM domain, is known to form homo or heterocomplexes that modulate diverse biological functions through the regulation of specific protein-protein interactions. The MAPK pathway of budding yeast Saccharomyces cerevisiae is comprised of a three-tier kinase system akin to mammals. The MAPKKK Ste11 protein of yeast contains a homodimer SAM domain, which is critical for transmitting cues to the downstream kinases. The structural stability of the dimeric Ste11 SAM is maintained by hydrophobic and ionic interactions at the interfacial amino acids. The urea-induced equilibrium-unfolding process of the Ste11 SAM domain is cooperative without evidence of any intermediate states. The native-state H/D exchange under subdenaturing conditions is a useful method for the detection of intermediate states of proteins. In the present study, we investigated the effect of ionic strength on the conformational stability of the dimer using the H/D exchange experiments. The hydrogen exchange behavior of the Ste11 dimer under physiological salt concentrations reveals two partially unfolded metastable intermediate states, which may be generated by a sequential and cooperative unfolding of the five helices present in the domain. These intermediates appear to be significant for the reversible unfolding kinetics via hydrophobic collapse. In contrast, higher ionic concentrations eliminate this cooperative interactions that stabilize the pairs of helices.

Design, Engineering and Discovery of Novel α-Helical and ß-Boomerang Antimicrobial Peptides against Drug Resistant Bacteria.

In an era where the pipeline of new antibiotic development is drying up, the continuous rise of multi-drug resistant (MDR) and extensively drug resistant (XDR) bacteria are genuine threats to human health. Although antimicrobial peptides (AMPs) may serve as promising leads against drug resistant bacteria, only a few AMPs are in advanced clinical trials. The limitations of AMPs, namely their low in vivo activity, toxicity, and poor bioavailability, need to be addressed. Here, we review engineering of frog derived short α-helical AMPs (aurein, temporins) and lipopolysaccharide (LPS) binding designed ß-boomerang AMPs for further development. The discovery of novel cell selective AMPs from the human proprotein convertase furin is also discussed.

Special Issue "Selected Papers from the 8th Asia-Pacific NMR (APNMR) Symposium: Recent Advances in NMR Spectroscopy".

Asia-Pacific NMR (APNMR) has been an important scientific event in the region, engaging a large number of NMR scientists from academia and industries [...].

NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion.

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15N{1H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV.

Elucidating the bactericidal mechanism of action of the linear antimicrobial tetrapeptide BRBR-NH2.

Linear antimicrobial peptides, with their rapid bactericidal mode of action, are well-suited for development as topical antibacterial drugs. We recently designed a synthetic linear 4-residue peptide, BRBR-NH2, with potent bactericidal activity against Staphylococcus aureus (MIC 6.25 µM), the main causative pathogen of human skin infections with an unknown mechanism of action. Herein, we describe a series of experiments conducted to gain further insights into its mechanism of action involving electron microscopy, artificial membrane dye leakage, solution- and solid-state NMR spectroscopy followed by molecular dynamics simulations. Experimental results point towards a SMART (Soft Membranes Adapt and Respond, also Transiently) mechanism of action, suggesting that the peptide can be developed as a topical antibacterial agent for treating drug-resistant Staphylococcus aureus infections.

'Lollipop'-shaped helical structure of a hybrid antimicrobial peptide of temporin B-lipopolysaccharide binding motif and mapping cationic residues in antibacterial activity.

BACKGROUND: Temporins are attractive templates for the development of antibiotics. However, many temporins are inactive against Gram-negative bacteria. Previously, we demonstrated conjugation of a lipopolysaccharide binding motif peptide to temporins yielded hybrid non-haemolytic AMPs that killed several Gram-negative bacteria. METHODS: We carried out a systematic Ala replacement of individual cationic and polar amino acid residues of LG21, a hybrid AMP consisted of temporin B (TB) and LPS binding motif. These Ala containing analogs of LG21 were examined for antibacterial activity, cell membrane permeabilization and liposome leakage assays using optical spectroscopic methods. Atomic resolution structure of LG21 was determined in zwitterionic dodecyl phosphocholine (DPC) micelles by NMR spectroscopy. RESULTS: Cationic residues in the LPS binding motif of LG21 were critical for bactericidal and membrane permeabilization. Detergent bound structure of LG21 revealed helical conformation containing extensive sidechain/sidechain packing including cation/π interactions in the LPS binding motif. The helical structure of LG21 resembled a 'lollipop' like shape that was sustained by a compacted bulky aromatic/cationic head with a comparatively thinner 'stick' at the N-terminal region. The 'head' of the structure could be localized into micelle-water interfacial region whereas the 'stick' region may be inserted into the hydrophobic core of micelle. CONCLUSIONS: The LPS binding motif of LG21 played dominant roles in broad spectrum activity and the 3-D structure provided plausible mechanistic insights for permeabilization of bacterial membrane. GENERAL SIGNIFICANCE: Hybrid AMPs containing LPS binding motif could be useful for the structure based development of broad spectrum antibiotics.

Designed Heme-Cage ß-Sheet Miniproteins.

The structure and function of naturally occurring proteins are governed by a large number of amino acids (≥100). The design of miniature proteins with desired structures and functions not only substantiates our knowledge about proteins but can also contribute to the development of novel applications. Excellent progress has been made towards the design of helical proteins with diverse functions. However, the development of functional ß-sheet proteins remains challenging. Herein, we describe the construction and characterization of four-stranded ß-sheet miniproteins made up of about 19 amino acids that bind heme inside a hydrophobic binding pocket or "heme cage" by bis-histidine coordination in an aqueous environment. The designed miniproteins bound to heme with high affinity comparable to that of native heme proteins. Atomic-resolution structures confirmed the presence of a four-stranded ß-sheet fold. The heme-protein complexes also exhibited high stability against thermal and chaotrope-induced unfolding.

NMR structures and localization of the potential fusion peptides and the pre-transmembrane region of SARS-CoV: Implications in membrane fusion.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) poses a serious public health hazard. The S2 subunit of the S glycoprotein of SARS-CoV carries out fusion between the virus and the host cells. However, the exact mechanism of the cell fusion process is not well understood. Current model suggests that a conformational transition, upon receptor recognition, of the two heptad core regions of S2 may expose the hydrophobic fusogenic peptide or fusion peptide for membrane insertion. Three regions of the S2 subunit have been proposed to be involved in cell-cell fusion. The N-terminal fusion peptide (FP, residues 770-788), an internal fusion peptide (IFP, residues 873-888) and the pre-transmembrane region (PTM, residues 1185-1202) demonstrated interactions with model lipid membranes and potentially involved in the fusion process. Here, we have determined atomic resolution structures of these three peptides in DPC detergent micelles by solution NMR. FP assumes α-helical conformation with significant distortion at the central Gly residues; enabling a close packing among sidechains of aromatic residues including W, Y and F. The 3-D structure of PMT is characterized by a helix-loop-helix with extensive aromatic interactions within the helices. IFP adopts a rather straight α-helical conformation defined by packing among sidechains of aromatic and aliphatic residues. Paramagnetic spin labeled NMR has demonstrated surface localization of PMT whereas FP and IFP inserted into the micelles. Collectively, data presented in this study will aid in understanding fusion mechanism of SARS-CoV.

Salt-resistant short antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising leads for the development of antibiotics against drug resistant bacterial pathogens. However, in vivo applications of AMPs remain obscure due to salt and serum mediated inactivation. The high cost of chemical synthesis of AMPs also impedes potential clinical application. Consequently, short AMPs resistant toward salt and serum inactivation are desirable for the development of peptide antibiotics. In this work, we designed a 12-residue amphipathic helical peptide RR12 (R-R-L-I-R-L-I-L-R-L-L-R-amide) and two Trp containing analogs of RR12 namely RR12Wpolar (R-R-L-I-W-L-I-L-R-L-L-R-amide), and RR12Whydro (R-R-L-I-R-L-W-L-R-L-L-R-amide). Designed peptides demonstrated potent antibacterial activity; MIC ranging from 2 to 8 µM, in the presence of sodium chloride (150 mM and 300 mM). Antibacterial activity of these peptides was also detected in the presence of human serum. Designed peptides, in particular RR12 and RR12Whydro, were only poorly hemolytic. As a mode of action; these peptides demonstrated efficient permeabilization of bacterial cell membrane and lysis of cell structure. We further investigated interactions of the designed peptides with lipopolysaccharide (LPS), the major component of the outer membrane permeability barrier of Gram-negative bacteria. Designed peptides adopted helical conformations in complex with LPS. Binding of peptides with LPS has yielded dissociation the aggregated structures of LPS. Collectively, these designed peptides hold ability to be developed for salt-resistant antimicrobial compounds. Most importantly, current work provides insights for designing salt-resistant antimicrobial peptides. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 345-356, 2016.

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

BACKGROUND: Protegin-1 (PG-1: RGGRLCYCRRRFCVCVGR-amide) assumes a rigid ß-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 ß-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 ß-hairpin like structure in complex with LPS. The ß-hairpin structure may be essential for outer membrane permeabilization and cell killing.

Characterization of the near native conformational states of the SAM domain of Ste11 protein by NMR spectroscopy.

The sterile alpha motif or SAM domain is one of the most frequently present protein interaction modules with diverse functional attributions. SAM domain of the Ste11 protein of budding yeast plays important roles in mitogen-activated protein kinase cascades. In the current study, urea-induced, at subdenaturing concentrations, structural, and dynamical changes in the Ste11 SAM domain have been investigated by nuclear magnetic resonance spectroscopy. Our study revealed that a number of residues from Helix 1 and Helix 5 of the Ste11 SAM domain display plausible alternate conformational states and largest chemical shift perturbations at low urea concentrations. Amide proton (H/D) exchange experiments indicated that Helix 1, loop, and Helix 5 become more susceptible to solvent exchange with increased concentrations of urea. Notably, Helix 1 and Helix 5 are directly involved in binding interactions of the Ste11 SAM domain. Our data further demonstrate that the existence of alternate conformational states around the regions involved in dimeric interactions in native or near native conditions.