scholarly journals The Central PXXP Motif Is Crucial for PMAP-23 Translocation across the Lipid Bilayer

2021 ◽  
Vol 22 (18) ◽  
pp. 9752
Author(s):  
Sung-Tae Yang ◽  
Song-Yub Shin ◽  
Sung-Heui Shin
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Membrane Surface ◽  
Helical Structure ◽  
Bacterial Cells ◽  
Bacterial Membranes ◽  
Pxxp Motif ◽  
Host Defense Peptide ◽  
Α Helix ◽  
Intracellular Targets

PMAP-23, a cathelicidin-derived host defense peptide, does not cause severe membrane permeabilization, but exerts strong and broad-spectrum bactericidal activity. We have previously shown that it forms an amphipathic α-helical structure with a central hinge induced by the PXXP motif, which is implicated in the interaction of PMAP-23 with negatively charged bacterial membranes. Here, we studied the potential roles of the PXXP motif in PMAP-23 translocation across the lipid bilayer by replacing Pro residues with either α-helix former Ala (PMAP-PA) or α-helix breaker Gly (PMAP-PG). Although both PMAP-PA and PMAP-PG led to effective membrane depolarization and permeabilization, they showed less antimicrobial activity than wild-type PMAP-23. Interestingly, we observed that PMAP-23 crossed lipid bilayers much more efficiently than its Pro-substituted derivatives. The fact that the Gly-induced hinge was unable to replace the PXXP motif in PMAP-23 translocation suggests that the PXXP motif has unique structural properties other than the central hinge. Surface plasmon resonance sensorgrams showed that the running buffer almost entirely dissociated PMAP-23 from the membrane surface, while its Pro-substituted derivatives remained significantly bound to the membrane. In addition, kinetic analysis of the sensorgrams revealed that the central PXXP motif allows PMAP-23 to rapidly translocate at the interface between the hydrophilic and hydrophobic phases. Taken together, we propose that the structural and kinetic understanding of the PXXP motif in peptide translocation could greatly aid the development of novel antimicrobial peptides with intracellular targets by promoting peptide entry into bacterial cells.

scholarly journals Interactions between the plasma membrane and the antimicrobial peptide HP (2-20) and its analogues derived from Helicobacter pylori

2006 ◽  
Vol 394 (1) ◽  
pp. 105-114 ◽  
Author(s):  
Kwang H. Lee ◽  
Dong G. Lee ◽  
Yoonkyung Park ◽  
Dong-Il Kang ◽  
Song Y. Shin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Helicobacter Pylori ◽  
Membrane Surface ◽  
Helical Structure ◽  
Spin Labels ◽  
Haemolytic Activity ◽  
Bacterial Cells ◽  
New Antibiotics ◽  
Bacteria And Fungi ◽  
Α Helix

HP (2-20), a 19-residue peptide derived from the N-terminus of Helicobacter pylori ribosomal protein L1, has antimicrobial activity but is not cytotoxic to human erythrocytes. We synthesized several peptide analogues to investigate the effects of substitutions on structure and antimicrobial activity. Replacement of Gln16 and Asp18 with tryptophan [anal-3 (analogue-3)] caused a dramatic increase in lytic activities against bacteria and fungi. By contrast, a decrease in amphiphilicity caused by replacement of Phe5 or Leu11 with serine was accompanied by a reduction in antimicrobial activity. Analysis of the tertiary structures of the peptides in SDS micelles by NMR spectroscopy revealed that they have a well-defined α-helical structure. Among the analogues, anal-3 has the longest α-helix, from Val4 to Trp18. The enhanced hydrophobicity and increased α-helicity results in enhanced antimicrobial activity in anal-3 without an increase in haemolytic activity. Fluorescence experiments proved that the bacterial-cell selectivity of the anal-3 peptide is due to its high binding affinity for negatively charged phospholipids in bacterial cells. Results showing the effect of spin-labels on the NMR spectra indicated that the side chains in the hydrophobic phase of the amphiphilic α-helix are buried on the surface of the micelle and the tryptophan indole ring is anchored in the membrane surface. Because anal-3 shows high selectivity towards bacterial and fungal cells, it may provide an avenue for the development of new antibiotics.

scholarly journals Non-Lytic Antibacterial Peptides That Translocate Through Bacterial Membranes to Act on Intracellular Targets

2019 ◽  
Vol 20 (19) ◽  
pp. 4877 ◽  
Author(s):  
Marlon H. Cardoso ◽  
Beatriz T. Meneguetti ◽  
Bruna O. Costa ◽  
Danieli F. Buccini ◽  
Karen G. N. Oshiro ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Synthesis ◽  
Pathogenic Bacteria ◽  
Biological Properties ◽  
Antibacterial Peptides ◽  
Bacterial Cells ◽  
Bacterial Membranes ◽  
Intracellular Targets

The advent of multidrug resistance among pathogenic bacteria has attracted great attention worldwide. As a response to this growing challenge, diverse studies have focused on the development of novel anti-infective therapies, including antimicrobial peptides (AMPs). The biological properties of this class of antimicrobials have been thoroughly investigated, and membranolytic activities are the most reported mechanisms by which AMPs kill bacteria. Nevertheless, an increasing number of works have pointed to a different direction, in which AMPs are seen to be capable of displaying non-lytic modes of action by internalizing bacterial cells. In this context, this review focused on the description of the in vitro and in vivo antibacterial and antibiofilm activities of non-lytic AMPs, including indolicidin, buforin II PR-39, bactenecins, apidaecin, and drosocin, also shedding light on how AMPs interact with and further translocate through bacterial membranes to act on intracellular targets, including DNA, RNA, cell wall and protein synthesis.

scholarly journals Membrane Structures of the Hemifusion-Inducing Fusion Peptide Mutant G1S and theFusion-Blocking Mutant G1V of Influenza Virus HemagglutininSuggest a Mechanism for Pore Opening in MembraneFusion

2005 ◽  
Vol 79 (18) ◽  
pp. 12065-12076 ◽  
Author(s):  
Yinling Li ◽  
Xing Han ◽  
Alex L. Lai ◽  
John H. Bushweller ◽  
David S. Cafiso ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Lipid Bilayer ◽  
Membrane Surface ◽  
Helical Structure ◽  
Fusion Peptide ◽  
Membrane Structures ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Pore Opening

ABSTRACT Influenza virus hemagglutinin (HA)-mediated membrane fusion is initiated by a conformational change that releases a V-shaped hydrophobic fusion domain, the fusion peptide, into the lipid bilayer of the target membrane. The most N-terminal residue of this domain, a glycine, is highly conserved and is particularly critical for HA function; G1S and G1V mutant HAs cause hemifusion and abolish fusion, respectively. We have determined the atomic resolution structures of the G1S and G1V mutant fusion domains in membrane environments. G1S forms a V with a disrupted “glycine edge” on its N-terminal arm and G1V adopts a slightly tilted linear helical structure in membranes. Abolishment of the kink in G1V results in reduced hydrophobic penetration of the lipid bilayer and an increased propensity to formβ -structures at the membrane surface. These results underline the functional importance of the kink in the fusion peptide and suggest a structural role for the N-terminal glycine ridge in viral membrane fusion.

scholarly journals The Human LL-37(17-29) Antimicrobial Peptide Reveals a Functional Supramolecular Nanostructure

2020 ◽  
Author(s):  
Yizhaq Engelberg ◽  
Meytal Landau
Keyword(s):  
Lipid Bilayers ◽  
Self Assembly ◽  
Amyloid Fibrils ◽  
Biological Activities ◽  
Building Blocks ◽  
Bacterial Cells ◽  
Bacterial Membranes ◽  
Self Assembling ◽  
Wide Ribbon ◽  
Wide Range

Protein fibrils that perform biological activities present attractive biomaterials. Here we demonstrate, by crystal structures, the self-assembly of the antibacterial human LL-37 active core (residues 17-29) into a stable structure of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-3717-29 correspondingly formed wide, ribbon-like, thermostable fibrils in solution, which co-localized with bacterial cells, and structure-guided mutagenesis analyses supported the role of self-assembly in antibacterial activity. LL-3717-29 resembled, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This suggests helical, self-assembling, basic building blocks across kingdoms of life and point to potential structural mimicry mechanisms. The findings offer a scaffold for functional and durable nanostructures for a wide range of medical and technological applications.

scholarly journals Lipid bilayer composition modulates the unfolding free energy of a knotted α-helical membrane protein

2018 ◽  
Vol 115 (8) ◽  
pp. E1799-E1808 ◽  
Author(s):  
M. R. Sanders ◽  
H. E. Findlay ◽  
P. J. Booth
Keyword(s):  
Free Energy ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Thermodynamic Stability ◽  
Lateral Pressure ◽  
Helical Structure ◽  
Free Energies ◽  
Transport Activity ◽  
Helical Protein ◽  
Urea Unfolding

α-Helical membrane proteins have eluded investigation of their thermodynamic stability in lipid bilayers. Reversible denaturation curves have enabled some headway in determining unfolding free energies. However, these parameters have been limited to detergent micelles or lipid bicelles, which do not possess the same mechanical properties as lipid bilayers that comprise the basis of natural membranes. We establish reversible unfolding of the membrane transporter LeuT in lipid bilayers, enabling the comparison of apparent unfolding free energies in different lipid compositions. LeuT is a bacterial ortholog of neurotransmitter transporters and contains a knot within its 12-transmembrane helical structure. Urea is used as a denaturant for LeuT in proteoliposomes, resulting in the loss of up to 30% helical structure depending upon the lipid bilayer composition. Urea unfolding of LeuT in liposomes is reversible, with refolding in the bilayer recovering the original helical structure and transport activity. A linear dependence of the unfolding free energy on urea concentration enables the free energy to be extrapolated to zero denaturant. Increasing lipid headgroup charge or chain lateral pressure increases the thermodynamic stability of LeuT. The mechanical and charge properties of the bilayer also affect the ability of urea to denature the protein. Thus, we not only gain insight to the long–sought-after thermodynamic stability of an α-helical protein in a lipid bilayer but also provide a basis for studies of the folding of knotted proteins in a membrane environment.

scholarly journals Pse-T2, an Antimicrobial Peptide with High-Level, Broad-Spectrum Antimicrobial Potency and Skin Biocompatibility against Multidrug-ResistantPseudomonas aeruginosaInfection

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
Hee Kyoung Kang ◽  
Chang Ho Seo ◽  
Tudor Luchian ◽  
Yoonkyung Park
Keyword(s):  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Helical Structure ◽  
Multidrug Resistant ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Bacterial Membranes

ABSTRACTPseudin-2, isolated from the frogPseudis paradoxa, exhibits potent antibacterial activity but also cytotoxicity. In an effort to develop clinically applicable antimicrobial peptides (AMPs), we designed pseudin-2 analogs with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. In addition, truncated analogs of pseudin-2 and Lys-substituted peptides were synthesized to produce linear 18-residue amphipathic α-helices, which were further investigated for their mechanism and functions. These truncated analogs exhibited higher antimicrobial activity and lower cytotoxicity than pseudin-2. In particular, Pse-T2 showed marked pore formation, permeabilization of the outer/inner bacterial membranes, and DNA binding. Fluorescence spectroscopy and scanning electron microscopy showed that Pse-T2 kills bacterial cells by disrupting membrane integrity.In vivo, wounds infected with multidrug-resistant (MDR)Pseudomonas aeruginosahealed significantly faster when treated with Pse-T2 than did untreated wounds or wounds treated with ciprofloxacin. Moreover, Pse-T2 facilitated infected-wound closure by reducing inflammation through suppression of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α). These data suggest that the small antimicrobial peptide Pse-T2 could be useful for future development of therapeutic agents effective against MDR bacterial strains.

COMPUTER SIMULATIONS OF MEMBRANE-LYTIC PEPTIDES: PERSPECTIVES IN DRUG DESIGN

2007 ◽  
Vol 05 (02b) ◽  
pp. 611-626 ◽  
Author(s):  
ANTON A. POLYANSKY ◽  
PAVEL E. VOLYNSKY ◽  
ROMAN G. EFREMOV
Keyword(s):  
Lipid Bilayers ◽  
Contact Region ◽  
Membrane Surface ◽  
Sodium Dodecyl ◽  
Md Simulations ◽  
Model Membranes ◽  
Sodium Dodecyl Sulfate Micelle ◽  
Bacterial Membranes ◽  
Local Character ◽  
Lytic Peptides

Structure activity relationships were investigated for membrane-lytic peptides (MLP) Ltc1 and Ltc2a from the latarcin family. The peptides were studied via long-term molecular dynamics (MD) simulations in different membrane environments (detergent micelles, mixed lipid bilayers mimiking eukaryotic and bacterial membranes). The calculated structure of Ltc2a in sodium dodecyl sulfate micelle agrees well with the data obtained by 1H-NMR spectroscopy. This validates the applied modeling approach. The binding mode of MLPs is governed by several factors: (i) the membrane surface curvature; (ii) the conformational plasticity and hydrophobic organization of the peptide, which depend on the arrangement of charged, non-polar and helix-breaking residues in the amino acid sequence. In contrast to Ltc1, insertion of Ltc2a into model membranes induces significant changes in dynamic behavior of lipids in the contact region. Such a prominent membrane destabilization correlates with high membrane-lytic activity of Ltc2a. In all cases the "membrane response" has a local character and is caused by formation of specific peptide-lipid contacts. Results of MD simulations of Ltc2a in model membranes were used to develop a number of its analogs with predefined activity.

scholarly journals A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes

2021 ◽  
Author(s):  
Mónica Gutiérrez-Salazar ◽  
Eduardo Santamaría-Aranda ◽  
Louise Schaar ◽  
Jesús Salgado ◽  
Diego Sampedro ◽  
...  
Keyword(s):  
Ftir Spectroscopy ◽  
Lipid Bilayers ◽  
Blue Light ◽  
Uv Light ◽  
Helical Structure ◽  
Membrane Topology ◽  
Step Model ◽  
Azobenzene Moiety ◽  
Helical Peptide ◽  
Α Helix

AbstractAccording to the three-step model, the spontaneous insertion and folding of helical transmembrane (TM) polypeptides into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion. However, understanding these three steps with molecular detail has been challenged by the lack of suitable experimental approaches to rapidly and reversibly perturb membrane-bound hydrophobic polypeptides out of equilibrium. Here, we report on a 24-residues-long hydrophobic α-helical polypeptide, covalently coupled to an azobenzene photoswitch (KCALP-azo), which displays a light-controllable TM/MI equilibrium in hydrated lipid bilayers. FTIR spectroscopy shows that dark-adapted KCALP-azo (trans azobenzene) folds as a TM α-helix, with its central TM region displaying an average tilt of 36 ± 4° with the membrane normal (TM topology). After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), spectral changes by FTIR spectroscopy indicate that the helical structure of KCALP-azo is maintained but the peptide experiences a more polar environment. Interestingly, pH changes induced similar spectral alterations in the helical peptide LAH4, with a well-characterized pH-dependent TM/MI equilibrium. Polarized experiments confirmed that the membrane topology of KCALP-azo is altered by light, with its helix tilt changing reversibly from 32 ± 5° (TM topology, blue light) to 79 ± 8° (MI topology, UV light). Further analysis indicates that, while the trans isomer of KCALP-azo is ~100% TM, the cis isomer exists in a ~90% TM and ~10% MI mixture. Strategies to further increase the perturbation of the TM/MI equilibrium with the light are briefly discussed.

scholarly journals Overlapping Properties of the Short Membrane-Active Peptide BP100 With (i) Polycationic TAT and (ii) α-helical Magainin Family Peptides

2021 ◽  
Vol 11 ◽  
Author(s):  
Christian Mink ◽  
Erik Strandberg ◽  
Parvesh Wadhwani ◽  
Manuel N. Melo ◽  
Johannes Reichert ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Mechanism Of Action ◽  
Helical Structure ◽  
Lipid Vesicles ◽  
Cell Penetrating Peptides ◽  
Cell Uptake ◽  
Active Peptide ◽  
Hiv Tat ◽  
Cell Penetrating ◽  
Α Helix

BP100 is a short, designer-made membrane-active peptide with multiple functionalities: antimicrobial, cell-penetrating, and fusogenic. Consisting of five lysines and 6 hydrophobic residues, BP100 was shown to bind to lipid bilayers as an amphipathic α-helix, but its mechanism of action remains unclear. With these features, BP100 embodies the characteristics of two distinctly different classes of membrane-active peptides, which have been studied in detail and where the mechanism of action is better understood. On the one hand, its amphiphilic helical structure is similar to the pore forming magainin family of antimicrobial peptides, though BP100 is much too short to span the membrane. On the other hand, its length and high charge density are reminiscent of the HIV-TAT family of cell penetrating peptides, for which inverted micelles have been postulated as translocation intermediates, amongst other mechanisms. Assays were performed to test the antimicrobial and hemolytic activity, the induced leakage and fusion of lipid vesicles, and cell uptake. From these results the functional profiles of BP100, HIV-TAT, and the magainin-like peptides magainin 2, PGLa, MSI-103, and MAP were determined and compared. It is observed that the activity of BP100 resembles most closely the much longer amphipathic α-helical magainin-like peptides, with high antimicrobial activity along with considerable fusogenic and hemolytic effects. In contrast, HIV-TAT shows almost no antimicrobial, fusogenic, or hemolytic effects. We conclude that the amphipathic helix of BP100 has a similar membrane-based activity as magainin-like peptides and may have a similar mechanism of action.

scholarly journals Naphthalimide-Containing BP100 Leads to Higher Model Membranes Interactions and Antimicrobial Activity

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 542
Author(s):  
Gustavo Penteado Battesini Carretero ◽  
Greice Kelle Viegas Saraiva ◽  
Magali Aparecida Rodrigues ◽  
Sumika Kiyota ◽  
Marcelo Porto Bemquerer ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Biological Activities ◽  
Biological Effects ◽  
Membrane Surface ◽  
Helical Structure ◽  
Helical Conformation ◽  
Property A ◽  
Cellular Processes ◽  
Double Stranded Dna ◽  
Low Toxicity

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm.

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