How Insertion of a Single Tryptophan in the N-Terminus of a Cecropin A-Melittin Hybrid Peptide Changes Its Antimicrobial and Biophysical Profile

In the era of antibiotic resistance, there is an urgent need for efficient antibiotic therapies to fight bacterial infections. Cationic antimicrobial peptides (CAMP) are promising lead compounds given their membrane-targeted mechanism of action, and high affinity towards the anionic composition of bacterial membranes. We present a new CAMP, W-BP100, derived from the highly active BP100, holding an additional tryptophan at the N-terminus. W-BP100 showed a broader antibacterial activity, demonstrating a potent activity against Gram-positive strains. Revealing a high partition constant towards anionic over zwitterionic large unilamellar vesicles and inducing membrane saturation at a high peptide/lipid ratio, W-BP100 has a preferential location for hydrophobic environments. Contrary to BP100, almost no aggregation of anionic vesicles is observed around saturation conditions and at higher concentrations no aggregation is observed. With these results, it is possible to state that with the incorporation of a single tryptophan to the N-terminus, a highly active peptide was obtained due to the π–electron system of tryptophan, resulting in negatively charged clouds, that participate in cation–π interactions with lysine residues. Furthermore, we propose that W-BP100 action can be achieved by electrostatic interactions followed by peptide translocation.

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Abstract 396: Acrolein Modification Weakens the Antimicrobial Activity of Apolipoprotein A1

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.396 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Paul Weers ◽

Hea J Hong

Keyword(s):

Antimicrobial Activity ◽

Electrostatic Interactions ◽

Structural Changes ◽

Apolipoprotein A1 ◽

Molar Ratio ◽

Antimicrobial Protein ◽

Anionic Phospholipid ◽

Protein Ratio ◽

Bacterial Membranes ◽

Lysine Residues

Human apolipoprotein A-I (apoA-I) has been show to exhibit antimicrobial activity by neutralizing lipopolysaccharides and destabilizing inner membranes of gram-negative bacteria. Previous studies showed that acrolein, a highly reactive αβ unsaturated aldehyde generated in cigarette smoking, modifies ε-amino side chains of lysine residues in apoA-I. The current study investigated the effect of acrolein exposure on the structure and antimicrobial activity of apoA-I. Incubation of apoA-I with acrolein using a 1:20 molar ratio, acrolein modification was evident by the appearance of apoA-I oligomers due to intermolecular crosslinking. Increase of the acrolein to protein ratio resulted in heavily cross-linked apoA-I, with protein bands appearing at 63, 98, and 126 kDa. The presence of acrolein-modified lysines in the oligomers was verified through Western blot analysis using mab5F6 antibody that specifically detects acrolein modified lysine residues in proteins. The structural changes of modified apoA-I was analyzed using circular dichroism. The α-helical content of acrolein-modified apoA-I was not significantly different from the unmodified protein. However, the midpoint of guanidine-induced denaturation increased from 0.97 to 1.50 M guanidine upon modification, indicating a significant increase in protein stability. This suggests that while modification did not alter the secondary structure, the protein fold was altered due to cross-linking. To measure the effect of acrolein modification on the interaction with bacterial membranes, binding experiments were performed with phosphatidylglycerol entrapped with calcein. This showed that the percentage of calcein released by apoA-I decreased from 87.5 ± 2.3 % to 4.7 ± 0.13 % when the protein was modified by acrolein. Thus acrolein-modified apoA-I binds phosphatidylglycerol less effectively, possibly due to loss of electrostatic interactions with anionic phospholipid vesicles. In addition, binding of apoA-I to lipopolysaccharides was significantly weaker when the protein was modified by acrolein. These results suggest that apoA-I modification by acrolein results in a protein with a decreased ability to bind to bacterial membranes and is thus less potent as an antimicrobial protein.

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Disruption of Membrane by Colistin Kills Uropathogenic Escherichia coli Persisters and Enhances Killing of Other Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01481-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6867-6871 ◽

Cited By ~ 28

Author(s):

Peng Cui ◽

Hongxia Niu ◽

Wanliang Shi ◽

Shuo Zhang ◽

Hao Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Bacterial Infections ◽

Enhancement Effect ◽

Bacterial Cells ◽

Sulfa Drugs ◽

Content Type ◽

Bacterial Membranes ◽

Highly Active ◽

High Concentrations

ABSTRACTPersisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active againstEscherichia colipersisters at high concentrations (25 or 50 μg/ml). At a clinically relevant lower concentration (10 μg/ml), colistin alone had no apparent effect onE. colipersisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugsin vitro. The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.

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Characteristics of the Interaction between Thrombin Exosite1 and the Sequence 269-297 of Platelet Glycoprotein Ibα

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615193 ◽

1998 ◽

Vol 80 (08) ◽

pp. 310-315 ◽

Cited By ~ 8

Author(s):

Marie-Christine Bouton ◽

Christophe Thurieau ◽

Marie-Claude Guillin ◽

Martine Jandrot-Perrus

Keyword(s):

Platelet Activation ◽

Electrostatic Interactions ◽

Platelet Glycoprotein ◽

Thrombin Receptor ◽

Central Position ◽

Amidolytic Activity ◽

N Terminus ◽

Hydrolysis Of ◽

Chemical Cross Linking ◽

Positively Charged

SummaryThe interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbα and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbα to interact with thrombin. Three peptides were synthesized, including Ibα 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibα 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibα 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibα 269-287 and α-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when γ-thrombin was substituted for α-thrombin. Ibα 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with α-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibα 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibα 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

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Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

Biochemical Journal ◽

10.1042/bj20071436 ◽

2008 ◽

Vol 411 (3) ◽

pp. 523-530 ◽

Cited By ~ 12

Author(s):

Gary S. Laco ◽

Yves Pommier

Keyword(s):

Amino Acids ◽

Topoisomerase I ◽

Electrostatic Interactions ◽

Functional Domain ◽

Site Mutation ◽

Supercoiled Dna ◽

Terminal Domain ◽

Tryptophan Residues ◽

N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

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The C-terminal peptide of CCL21 drastically augments CCL21 activity through the dendritic cell lymph node homing receptor CCR7 by interaction with the receptor N-terminus

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-03930-7 ◽

2021 ◽

Author(s):

Astrid Sissel Jørgensen ◽

Emma Probst Brandum ◽

Jeppe Malthe Mikkelsen ◽

Klaudia A. Orfin ◽

Ditte Rahbæk Boilesen ◽

...

Keyword(s):

Lymph Node ◽

Electrostatic Interactions ◽

Immune Cell ◽

Direct Interaction ◽

Receptor Interaction ◽

Binding Model ◽

Lymph Node Homing ◽

Gating Mechanism ◽

N Terminus ◽

Positively Charged

AbstractThe endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initial chemokine binding, the receptor N-terminus also partakes in a gating mechanism, which could give rise to a reduced ligand activity, presumably through affecting the ligand positioning. Based on experiments that support a direct interaction of C21TP with the glycosylated CCR7 N-terminus, we propose that electrostatic interactions between the positively charged peptide and sialylated O-glycans in CCR7 N-terminus may create a more accessible version of the receptor and thus guide chemokine docking to generate a more favorable chemokine-receptor interaction, giving rise to the peptide boosting effect.

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The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro

European Journal of Biochemistry ◽

10.1046/j.1432-1327.2000.01366.x ◽

2000 ◽

Vol 267 (12) ◽

pp. 3461-3468 ◽

Cited By ~ 31

Author(s):

Xiao-Song Gong ◽

Jiang Qi Wen ◽

Nicholas E. Fisher ◽

Simon Young ◽

Christopher J. Howe ◽

...

Keyword(s):

Electrostatic Interactions ◽

Cytochrome F ◽

Lysine Residues

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Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

Molecular Biology of the Cell ◽

10.1091/mbc.e10-03-0256 ◽

2011 ◽

Vol 22 (2) ◽

pp. 189-201 ◽

Cited By ~ 40

Author(s):

Roman Gorelik ◽

Changsong Yang ◽

Vasumathi Kameswaran ◽

Roberto Dominguez ◽

Tatyana Svitkina

Keyword(s):

Plasma Membrane ◽

Electrostatic Interactions ◽

Coiled Coil ◽

Small Gtpases ◽

Coincidence Detection ◽

Membrane Targeting ◽

Amino Terminal ◽

N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

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GFP Fusion to the N-Terminus of MotB Affects the Proton Channel Activity of the Bacterial Flagellar Motor in Salmonella

Biomolecules ◽

10.3390/biom10091255 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1255

Author(s):

Yusuke V. Morimoto ◽

Keiichi Namba ◽

Tohru Minamino

Keyword(s):

Electrostatic Interactions ◽

Proton Translocation ◽

Proton Channel ◽

Flagellar Motor ◽

Wild Type ◽

Over Expression ◽

N Terminus ◽

Flow Through ◽

Proton Leakage ◽

Bacterial Flagellar Motor

The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.

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Antimicrobial and Antifungal Activities of Terpene-Derived Palladium Complexes

Antibiotics ◽

10.3390/antibiotics9050277 ◽

2020 ◽

Vol 9 (5) ◽

pp. 277 ◽

Cited By ~ 1

Author(s):

Olga Zalevskaya ◽

Yana Gur’eva ◽

Aleksandr Kutchin ◽

Karl A. Hansford

Keyword(s):

Staphylococcus Aureus ◽

Bacterial Infections ◽

Multidrug Resistant ◽

Palladium Complexes ◽

Antifungal Drugs ◽

Antifungal Activities ◽

Highly Active ◽

Development Pipeline ◽

Antimicrobial And Antifungal Activities ◽

Global Health Problem

In an era of multidrug-resistant bacterial infections overshadowed by a lack of innovation in the antimicrobial drug development pipeline, there has been a resurgence in multidisciplinary approaches aimed at tackling this global health problem. One such approach is to use metal complexes as a framework for new antimicrobials. Indeed, in this context, bismuth-, silver- and gold-derived compounds in particular have displayed demonstrable antimicrobial activity. In this work, we discuss the antimicrobial and antifungal activities of terpene-derived chiral palladium complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Candida albicans, and Cryptococcus neoformans. It was established that all studied coordination compounds of palladium were highly active antifungal drugs. In contrast, the subset of palladacycles possessing a palladium–carbon bond were only active against the Gram-positive bacterium Staphylococcus aureus. All compounds were inactive against the Gram-negative bacteria tested.

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A proline repeat domain in the Notch co-activator MAML1 is important for the p300-mediated acetylation of MAML1

Biochemical Journal ◽

10.1042/bj20061900 ◽

2007 ◽

Vol 404 (2) ◽

pp. 289-298 ◽

Cited By ~ 33

Author(s):

Mariana Saint Just Ribeiro ◽

Magnus L. Hansson ◽

Annika E. Wallberg

Keyword(s):

Target Genes ◽

Repeat Motif ◽

Terminal Domain ◽

Evolutionarily Conserved ◽

Repeat Domain ◽

Intracellular Receptor ◽

N Terminus ◽

Lysine Residues ◽

Receptor Domain

Ligand activation of Notch leads to the release of Notch IC (the intracellular receptor domain), which translocates to the nucleus and interacts with the DNA-binding protein CSL to control expression of specific target genes. In addition to ligand-mediated activation, Notch signalling can be further modulated by interactions of Notch IC with a number of other proteins. MAML1 has previously been shown to act co-operatively with the histone acetyltransferase p300 in Notch IC-mediated transcription. In the present study we show that the N-terminal domain of MAML1 directly interacts with both p300 and histones, and the p300–MAML1 complex specifically acetylates histone H3 and H4 tails in chromatin. Furthermore, p300 acetylates MAML1 and evolutionarily conserved lysine residues in the MAML1 N-terminus are direct substrates for p300-mediated acetylation. The N-terminal domain of MAML1 contains a proline repeat motif (PXPAAPAP) that was previously shown to be present in p53 and important for the p300–p53 interaction. We show that the MAML1 proline repeat motif interacts with p300 and enhances the activity of the MAML1 N-terminus in vivo. These findings suggest that the N-terminal domain of MAML1 plays an important role in Notch-regulated transcription, by direct interactions with Notch, p300 and histones.

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