The effect of a beta‐lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study

Co‐administration of beta‐lactam antibiotics and beta‐lactamase inhibitors has been a favored treatment strategy against beta‐lactamase‐mediated bacterial antibiotic resistance, but the emergence of beta‐lactamases resistant to current inhibitors necessitates the discovery of novel non‐beta‐lactam inhibitors. Peptides derived from the Ala46–Tyr51 region of the beta‐lactamase inhibitor protein are considered as potent inhibitors of beta‐lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell‐penetrating peptides could guide the design of beta‐lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta‐lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell‐penetrating peptide pVEC, our approach involved the addition of the N‐terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta‐lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta‐lactam antibiotic ampicillin, and the beta‐lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N‐terminus of the beta‐lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N‐terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Exploring the potential of boronic acids as inhibitors of OXA‐24/40 β‐lactamase

β‐lactam antibiotics are crucial to the management of bacterial infections in the medical community. Due to overuse and misuse, clinically significant bacteria are now resistant to many commercially available antibiotics. The most widespread resistance mechanism to β‐lactams is the expression of β‐lactamase enzymes. To overcome β‐lactamase mediated resistance, inhibitors were designed to inactivate these enzymes. However, current inhibitors (clavulanic acid, tazobactam, and sulbactam) for β‐lactamases also contain the characteristic β‐lactam ring, making them susceptible to resistance mechanisms employed by bacteria. This presents a critical need for novel, non‐β‐lactam inhibitors that can circumvent these resistance mechanisms. The carbapenem‐hydrolyzing class D β‐lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze potent carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available β‐lactamase inhibitors, nor are they effectively inhibited by the newest, non‐β‐lactam inhibitor, avibactam. Boronic acids are known transition state analog inhibitors of class A and C β‐lactamases, and are not extensively characterized as inhibitors of class D β‐lactamases. Importantly, boronic acids provide a novel way to potentially inhibit class D β‐lactamases. Sixteen boronic acids were selected and tested for inhibition of the CHDL OXA‐24/40. Several compounds were identified as effective inhibitors of OXA‐24/40, with K_i values as low as 5 μM. The X‐ray crystal structures of OXA‐24/40 in complex with BA3, BA4, BA8, and BA16 were determined and revealed the importance of interactions with hydrophobic residues Tyr112 and Trp115. These boronic acids serve as progenitors in optimization efforts of a novel series of inhibitors for class D β‐lactamases.     

Micelle bound structure and DNA interaction of brevinin‐2‐related peptide,an antimicrobial peptide derived from frog skin

Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

pVEC hydrophobic N‐terminus is critical for antibacterial activity

Cell‐penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure‐activity relationship and sequence alignment studies have suggested that the hydrophobic N‐terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N‐terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

The engineering of membrane-permeable peptides

Reversible lipid attachment was investigated as a means to deliver small peptides into cells. Two labile straight chain alkyl motifs were developed: a cysteine dodecane disulfide (Cdd) building block and a tyrosine- or serine-myristate ester. Both moieties are cleaved on cell internalization and are compatible with Fmoc solid phase peptide synthesis. A series of fluorophore-labeled peptides that varied in lipophilic content, net charge, and charge distribution were synthesized. The peptides were screened for cellular uptake efficiency as monitored by fluorescence microscopy. Effective peptide transport is based on a distributed net positive charge introduced as lysine residues at the C and/or N terminus of the peptide and the presence of a hydrophobic domain exhibiting an estimated log P4.0. The incorporation of labile lipid motifs into peptides enhances lipophilic character of the peptides and contributes to cellular uptake with minimal alteration to the native sequence.     

The agony of choice: how to find a suitable CPP for cargo delivery

Successful and effective cellular delivery remains a main obstacles in the medical field. The use of cell‐penetrating peptides (CPPs) has become one of the most important tools for the internalisation of a wide range of molecules including pharmaceuticals. It is still difficult to choose one CPP for one biological application because there is no ubiquitous CPP meeting the diverse requirements. In our case, we are looking for a suitable CPP to deliver the pro‐apoptotic KLA peptide (KLAKLAKKLAKLAK) by a simple co‐incubation strategy. For that reason, we selected three different cell lines (fibroblastic, cancerous and macrophagic cells) and studied the uptake and subcellular localisation of six different CPPs alone as well as mixed with the KLA peptide. Furthermore, we used the CPPs with a carboxyamidated or a carboxylated C‐terminus and analysed the impact of the C‐termini on internalisation and cargo delivery. We could clearly showed that the cellular CPP uptake is not only dependent on the used CPP and cell line but also highly affected by its chemical nature of the C‐terminus (uptake: carboxyamidated CPPs > carboxylated CPPs) and can influence its cellular localisation. We successfully delivered the KLA peptide in the three cell lines and learned that here as well, the C‐terminus is crucial for an effective peptide delivery. Finally, we induced apoptosis in mouse leukaemic monocyte macrophage (RAW 264.7) and in human breast adenocarcinoma (MCF‐7) cells using the mixture of amidated MPG peptide : KLA and in african green monkey kidney fibroblast (Cos‐7) cells using carboxylated integrin peptide : KLA. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Evaluation of lipid‐binding properties of the N‐terminal helical segments in human apolipoprotein A‐I using fragment peptides

Although the N‐terminal region in human apolipoprotein (apo) A‐I is thought to stabilize the lipid‐free structure of the protein, its role in lipid binding is unknown. Using synthetic fragment peptides, we examined the lipid‐binding properties of the first 43 residues (1–43) of apoA‐I in comparison with residues 44–65 and 220–241, which have strong lipid affinity in the molecule. Circular dichroism measurements demonstrated that peptides corresponding to each segment have potential propensity to form α‐helical structure in trifluoroethanol. Spectroscopic and thermodynamic measurements revealed that apoA‐I (1–43) peptide has the strong ability to bind to lipid vesicles and to form α‐helical structure comparable to apoA‐I (220–241) peptide. Substitution of Tyr‐18 located at the center of the most hydrophobic region in residues 1–43 with a helix‐breaking proline resulted in the impaired lipid binding, indicating that the α‐helical structure in this region is required to trigger the lipid binding. In contrast, apoA‐I (44–65) peptide exhibited a lower propensity to form α‐helical structure upon binding to lipid, and apoA‐I (44–65/S55P) peptide exhibited diminished, but not completely impaired, lipid binding, suggesting that the central region of residues 44–65 is not pivotally involved in the formation of the α‐helical structure and lipid binding. These results indicate that the most N‐terminal region of apoA‐I molecule, residues 1–43, contributes to the lipid interaction of apoA‐I through the hydrophobic helical residues. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Design of antimicrobial peptide arenicin analogs with improved therapeutic indices

β‐Hairpin antimicrobial peptides are among the most potent peptide antibiotics of animal origin. Arenicins, isolated earlier from marine polychaeta lugworm Arenicola marina, belong to a family of β‐hairpin antimicrobial peptides and display a broad spectrum of biological activities. However, despite being potent antimicrobials, arenicins are partially unapplicable as therapeutics as a result of their relatively high cytotoxicity against mammalian cells. In this study, a template‐based approach was used to create therapeutically valuable analogs of arenicin‐1 and identify amino acid residues important for antibacterial and cytotoxic activities of the peptide. The plasmids encoding recombinant analogs were constructed by mutagenesis technique based on inverse PCR amplification of the whole arenicin‐1 expression plasmid. The analogs were produced as a part of the fusion proteins in Escherichia coli. It was shown that an obvious reduction in hemolytic activity without lose of antimicrobial activity can be achieved by a single amino acid substitution in the non‐polar face of the molecule with hydrophilic residues such as serine and arginine. As the result, the selective analog with 50‐fold improved therapeutic index was developed. The circular dichroism spectra demonstrated that the secondary structure of the analog was similar to the natural arenicin‐1 in water solution and sodium dodecyl sulfate micelles but significantly differed in the presence of dodecylphosphocholine micelles mimicking mammalian membranes. Similarly to arenicin‐1, the designed analog killed bacteria via induction of the membrane damage, assessed using the fluorescent dye SYTOX Green uptake. Our results afford molecular insight into mechanism of antimicrobial action of the designed arenicin analogs and their possible clinical application. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide purification using the chemoselective reaction between N‐(methoxy)glycine and isothiocyanato‐functionalized resin

An efficient peptide purification strategy is established, comprising the selective reaction of an N‐terminal N‐(methoxy)glycine residue of the peptide and isothiocyanato‐functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation‐prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240–272). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Epidermoid carcinoma-derived antimicrobial peptide (ECAP) inhibits phosphorylation by protein kinases in vitro.

Animal peptide antibiotics are thought to mediate their cytotoxic and growth inhibitory action on bacteria, fungi, and cancer cells through a membrane-targeted mechanism. Although the membrane interactions of the peptide antibiotics and their penetration through the membranes have been studied in several models, the precise chain of events leading to cell death or growth arrest is not established yet. In this study we used in vitro kinase assays followed by imaging analyses to examine the effect of human cationic antimicrobial peptide ECAP on the activity of the protein kinases. We report that HPLC-grade ECAP is responsible for inhibition of EGFR autophosphorylation in plasma membrane fractions obtained from A-431 cells. The activity of ECAP is concentration dependent with a half-inhibitory concentration in the range of 0.1-0.2 microM. Marked decrease in autophosphorylation of immunoprecipitated non-receptor protein kinases belonging to different families, namely PKCmu, Lyn and Syk, is observed in the presence of as little as 0.2 microM of the peptide. Among the examined non-receptor protein kinases PKCmu was the most sensitive to the inhibitory action of ECAP, whereas Syk was inhibited least of all. ECAP exerted no detectable cytotoxicity on non-nucleate animal cells at concentrations up to 3 microM. The capability of ECAP to inhibit protein kinases at concentrations, that are at least 10 fold lower than antibacterial and cytotoxic ones, suggests that the protein kinases are possible intracellular targets for antimicrobial peptides. We suppose that inhibition of the protein kinases may provide a mechanism for the action of cationic antimicrobial peptides on host cells including tumour cells.     

Design,synthesis, and comparative evaluation of 99mTc(CO)3‐labeled N‐terminal and C‐terminal modified asparagine–glycine–arginine peptide constructs

The present study describes modification of asparagine–glycine–arginine (NGR) peptide at N‐terminally and C‐terminally by introduction of a tridentate chelating scaffold via click chemistry reaction. The N‐terminal and C‐terminal modified peptides were radiometalated with [^99mTc(CO)₃]⁺ precursor. The influence of these moieties at the two termini on the targeting properties of NGR peptide was determined by in vitro cell uptake studies and in vivo biodistribution studies. The two radiolabeled constructs did not exhibit any significant variation in uptake in murine melanoma B16F10 cells during in vitro studies. In vivo studies revealed nearly similar tumor uptake of N‐terminally modified peptide construct 5 and C‐terminally construct 6 at 2 h p.i. (1.9 ± 0.1 vs 2.4 ± 0.2% ID/g, respectively). The tumor‐to‐blood (T/B) and tumor‐to‐liver (T/L) ratios of the two radiometalated peptides were also quite similar. The two constructs cleared from all the major organs (heart, lungs, spleen, stomach, and blood) at 4 h p.i. (<1% ID/g). Blocking studies carried out by coinjection of cCNGRC peptide led to approximately 50% reduction in the tumor uptake at 2 h p.i. This work thus illustrates the possibility of convenient modification/radiometalation of NGR peptide at either N‐ or C‐terminus without hampering tumor targeting and pharmacokinetics.     

Antimicrobial activity of peptides derived from human ß‐amyloid precursor protein

Antimicrobial peptides are important effector molecules of the innate immune system. Here, we describe that peptides derived from the heparin‐binding disulfide‐constrained loop region of human ß‐amyloid precursor protein are antimicrobial. The peptides investigated were linear and cyclic forms of NWCKRGRKQCKTHPH (NWC15) as well as the cyclic form comprising the C‐terminal hydrophobic amino acid extension FVIPY (NWCKRGRKQCKTHPHFVIPY; NWC20c). Compared with the benchmark antimicrobial peptide LL‐37, these peptides efficiently killed the Gram‐negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram‐positive Staphylococcus aureus and Bacillus subtilis, and the fungi Candida albicans and Candida parapsilosis. Correspondingly, fluorescence and electron microscopy demonstrated that the peptides caused defects in bacterial membranes. Analogously, the peptides permeabilised negatively charged liposomes. Despite their bactericidal effect, the peptides displayed very limited hemolytic activities within the concentration range investigated and exerted very small membrane permeabilising effects on human epithelial cells. The efficiency of the peptides with respect to bacterial killing and liposome membrane leakage was in the order NWC20c > NWC15c > NWC15l, which also correlated to the adsorption density for these peptides at the model lipid membrane. Thus, whereas the cationic sequence is a minimum determinant for antimicrobial action, a constrained loop‐structure as well as a hydrophobic extension further contributes to membrane permeabilising activity of this region of amyloid precursor protein. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Engineering of a linear inactive analog of human β‐defensin 4 to generate peptides with potent antimicrobial activity

Human β‐defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N‐terminus of HBD4. Our results show that l ‐arginine to d ‐arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l ‐arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d ‐arginine. Substitution of cysteine with the hydrophobic helix‐promoting amino acid α‐aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d ‐arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d ‐amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibitory effect of short cationic homopeptides against Gram‐positive bacteria

In the selection or design of antimicrobial peptides, the key role played by cationic amino acids and chain length on the inhibitory potency and specificity is not clear. A fundamental study was conducted using chemically synthesized homopeptides of l ‐Lys and l ‐Arg ranging from 7 to 14 residues. Their effect on growth inhibition was evaluated over a wide range of Gram‐positive bacteria at different levels of concentration. Interestingly, at lower concentrations (10 μM), Lys homopeptides with odd number of residues, especially with 11 residues, showed a broader inhibitory activity than those with even number of residues. At higher peptide concentrations (>20 μM), the inhibitory activity of Lys homopeptides was directly related to the number of residues in the chain. In contrast, Arg homopeptides, at lower concentrations, did not exhibit a defined pattern of bacterial inhibition related to the number of residues; however, at higher concentrations (>20 μM), the inhibitory effects were more pronounced. Lys homopeptides at concentrations up to 300 μM showed a remarkably lower toxicity against CHSE‐214 cells. Arg homopeptides exhibited negligible cytotoxicity up to chain length of 11 residues at concentrations lower than 100 μM, but an abrupt increase in toxicity resulted when the peptide chain length reached 12 amino acid residues and higher concentrations. All synthesized homopeptides displayed characteristic polyproline II helix conformation in both buffer and liposomes, as shown by CD spectroscopy. This result suggests that short Lys homopeptides with an odd number of residues (9 and 11) have a broad spectrum of activity against Gram‐positive bacterial cells compared with Arg homopeptides, which in turn showed a considerably higher selectivity toward those cells. By investigating the differences between Lys and Arg homopeptides, this study contributes to the understanding of their mechanism of growth inhibition and selectivity. Thus, it provides further guidelines for a rational design of short antimicrobial peptides. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

SAR study and conformational analysis of a series of novel peptide G protein‐coupled receptor kinase 2 inhibitors

G protein‐coupled receptor kinase 2 (GRK2) plays a central role in the cellular transduction network. In particular, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. Thereby, its inhibition offers a potential therapeutic solution to several pathological conditions. In the present study, we performed a SAR study and a NMR conformational analysis of peptides derived from HJ loop of GRK2 and able to selectively inhibit GRK2. From Ala‐scan and d ‐Ala point replacement, we found that Arg residues don't affect the inhibitory properties, while a d ‐amino acid at position 5 is key to the activity. Conformational analysis identified two β‐turns that involve N‐terminal residues, followed by a short extended region. These information can help the design of peptides and peptido‐mimetics with enhanced GRK2 inhibition properties. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 121–128, 2014.     

Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions

Establishing a quantitative understanding of the determinants of affinity in protein–protein interactions remains challenging. For example, TEM‐1/β‐lactamase inhibitor protein (BLIP) and SHV‐1/BLIP are homologous β‐lactamase/β‐lactamase inhibitor protein complexes with disparate K_d values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV‐1, results in a 1000‐fold enhancement in binding affinity. In TEM‐1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV‐1 D104 does not in the wild type SHV‐1/BLIP co‐structure. Here, we present a 1.6 Å crystal structure of the SHV‐1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV‐1 D104 and BLIP K74 and a 400‐fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Structural basis for the β‐lactamase activity of EstU1, a family VIII carboxylesterase

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β‐lactam antibiotics as well as the ester bond of p‐nitrophenyl esters. EstU1 assumes a β‐lactamase‐like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β‐lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β‐lactam antibiotics. This result explains the weak β‐lactamase activity of EstU1 compared with class C β‐lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum. Proteins 2013; 81:2045–2051. © 2013 Wiley Periodicals, Inc.     

Modified Cysteine-Deleted Tachyplesin (CDT) Analogs as Linear Antimicrobial Peptides: Influence of Chain Length,Positive Charge,and Hydrophobicity on Antimicrobial and Hemolytic Activity

Due to increasing resistance of bacteria to traditional antibiotics, antimicrobial peptides are being investigated as a promising alternative. Tachyplesin, an antimicrobial peptide isolated from horseshoe crab, inhibits the growth of many different types of bacteria with its ability to permeabilize the cell membrane. Starting with a previously reported linear tachyplesin analog lacking cysteine (cysteine-deleted tachyplesin, CDT, KWFRVYRGIYRRR-CONH₂), this study examines the systematic deletion of the C-terminal arginines and the N-terminal lysine, addition of positively charged N-and C-terminal residues, replacement of arginine with similarly-charged lysine, and replacement of hydrophobic residues with aliphatic, aromatic, fluoro-substituted aromatic, and bicyclic amino acids to examine effects on activity. The 16 modified CDT analogs were tested for their ability to disrupt model liposomes, and minimum inhibitory concentrations were determined for gram-positive and gram-negative bacterial strains. Hemolytic activity also was assessed. Overall, results indicate that elimination of two C-terminal arginine residues results in a peptide ([des-Arg^12,13]CDT) with preserved antimicrobial activity but a reduction in hemolysis, a selectivity desirable for a therapeutic agent. Additional deletion was not tolerated, nor was addition of residues at the termini. Analysis of the 16 analogs also reveals the importance of hydrophobicity, not necessarily aromaticity, as an analog with hydrophobic isoleucine residues placed throughout the sequence ([Ile^2,3,6,10]CDT) displayed comparable antimicrobial activity to CDT with lower hemolysis, representing a promising antimicrobial peptide with lowered toxicity.     

Design,expression, and characterization of the hybrid antimicrobial peptide T‐catesbeianin‐1 based on FyuA

The overuse of antibiotics has resulted in the emergence of antibiotic‐resistant bacteria, which presents an urgent need for new antimicrobial agents. At present, antimicrobial peptides have attracted a great deal of attention from researchers. However, antimicrobial peptides often affect a broad range of microorganisms, including the normal flora in a host organism. In the present study, we designed a novel hybrid antimicrobial peptide, expressed the hybrid peptide, and studied its specific target. The hybrid peptide, named T‐catesbeianin‐1, which includes the FyuA‐binding domain of pesticin and the peptide catesbeianin‐1, was designed and expressed in Pichia pastoris X‐33. Then, we determined the antimicrobial activity, cytotoxicity, and specific target of the peptide. T‐catesbeianin‐1 has strong antimicrobial activity and binds to FyuA to inhibit or kill Escherichia coli present in clinical specimens and mixed‐species culture. In summary, these findings suggested that T‐catesbeianin‐1 might be promising and specific antibiotic agent for therapeutic application against fyuA⁺ E. coli.