Lysylated phospholipids stabilize models of bacterial lipid bilayers and protect against antimicrobial peptides

Aminoacylated phosphatidylglycerols are common lipids in bacterial cytoplasmic membranes. Their presence in Staphylococcus aureus has been linked to increased resistance to a number of antibacterial agents, including antimicrobial peptides. Most commonly, the phosphatidylglycerol headgroup is esterified to lysine, which converts anionic phosphatidylglycerol into a cationic lipid with a considerably increased headgroup size. In the present work, we investigated the interactions of two well-studied antimicrobial peptides, cecropin A and mastoparan X, with lipid vesicles composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), containing varying fractions of an aminoacylated phosphatidylethanolamine, a stable analog of the corresponding phosphatidylglycerol-derivative. To differentiate between the effects of headgroup size and charge on peptide–lipid interactions, we synthesized two different derivatives. In one, the headgroup was modified by the addition of lysine, and in the other, by glutamine. The modification by glutamine results in a phospholipid with a headgroup size comparable to that of the lysylated version. However, whereas lysyl-phosphatidylethanolamine (Lys-PE) is cationic, glutaminyl-phosphatidylethanolamine (Gln-PE) is zwitterionic. We found that binding of mastoparan X and cecropin A was not significantly altered if the content of aminoacylated phosphatidylethanolamines did not exceed 20 mol.%, which is the concentration found in bacterial membranes. However, a lysyl-phosphatidylethanolamine content of 20 mol% significantly inhibits dye release from lipid vesicles, to a degree that depends on the peptide. In the case of mastoparan X, dye release is essentially abolished at 20 mol.% lysyl-phosphatidylethanolamine, whereas cecropin A is less sensitive to the presence of lysyl-phosphatidylethanolamine. These observations are understood through the complex interplay between peptide binding and membrane stabilization as a function of the aminoacylated lipid content. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate

Escherichia coli outer membrane protein A (OmpA) is a well-established model for the study of membrane assembly. Previous studies have shown that the essential sequence for outer membrane localization, known as the sorting signal, is contained in a segment of the eighth β-strand, residues 163-171. Sequential digestion of OmpA, purified from outer membranes or inclusion bodies with cyanogen bromide and Staphylococcus aureus GluC, yielded peptides 162-174(LSLGVSYRFGQGE). Western blot and chemical assays indicated that the peptide was covalently modified by oligo-(R)-3-hydroxybutyrate (cOHB), a flexible, amphipathic oligoester. MALDI/MS was consistent with modification of peptides 162-174 by up to ten R-3-hydroxybutyrate (HB) residues. Western blot analysis of mutants of the peptide, using anti-OHB IgG, indicated that cOHB modification was not inhibited by the single mutations S163G, S167G, Y168F, R169N or R169D; however, cOHB was not detected on peptides containing the double mutations S163G:S167G S163G:V166G, L162G:S167G, and L164G:S167G. MALDI/MS/MS of double mutant S163G:S167G confirmed the absence of cOHB-modification. The results suggest that cOHB may be attached to one or both serines, and point to the importance of the flanking hydrophobic residues. Modification by cOHB may play a role in outer membrane targeting and assembly of OmpA.     

Effects of L- or D-Pro incorporation into hydrophobic or hydrophilic helix face of amphipathic alpha-helical model peptide on structure and cell selectivity

A synthetic amphipathic alpha-helical model peptide, KLW, displays non-cell selective cytotoxicity. To investigate the effects of L- or D-Pro kink incorporation into hydrophobic or hydrophilic helix face of KLW on structure, cell selectivity, and membrane-binding affinity, we designed a series of four peptides, in which Leu(9) and Lys(11) in the hydrophobic and hydrophilic helix face of KLW, respectively, are substituted with L- or D-Pro. A L- or D-Pro substitution (KLW-L9P or KLW-L9p) of Leu(9) at the hydrophobic helix face of KLW induced a more significant reduction in hemolytic activity with improved antibacterial activity than that (KLW-K11P or KLW-K11p) of Lys(11) in the hydrophilic helix face. In addition, D-Pro-containing peptides (KLW-L9p and KLW-K11p) displayed less hemolytic activity than L-Pro-containing peptides (KLW-L9P and KLW-K11P). Tryptophan fluorescence studies revealed that bacterial cell selectivity of KLW-L9P, KLW-L9p, and KLW-K11p is closely related to selective interactions with negatively charged phospholipids. CD analysis revealed that L- or D-Pro incorporation into KLW reduces the alpha-helicity of the peptide and D-Pro incorporation induces more significant disruption in alpha-helical structure than L-Pro incorporation. Our results collectively suggest that D-Pro incorporation into the hydrophobic helix face of non-cell selective amphipathic alpha-helical peptides may be useful for the design of novel antimicrobial peptides possessing high bacterial cell selectivity without hemolytic activity.     

Design of cyclic RGD-conjugated Aib-containing amphipathic helical peptides for targeted delivery of small interfering RNA

To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to α_Vβ₃ integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the α_Vβ₃ integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0 μM PI/10 nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA.     

Roles of Amphipathic Helices and the Bin/Amphiphysin/Rvs (BAR) Domain of Endophilin in Membrane Curvature Generation

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63–86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63–75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic α-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.     

NMR solution structure of the N-terminal domain of hERG and its interaction with the S4-S5 linker

The human Ether-à-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation. To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker.     

Synthesis of analogs of peptides from Buthus martensii scorpion venom with potential antibiotic activity

Five analogs of a natural peptide (BmKn1) found in the venom of scorpion Buthus martensii Karsh have been synthesized and tested to compare their antimicrobial and hemolytic activity with the wild type. Circular dichroism spectra show that these peptides form an alpha helix structure and its amino acid positions predict an amphipathic nature. Results show that increasing hydrophobicity by substituting successively positions 5 and 9 of the sequence (on the hydrophobic side of the helix) with alanine, valine and leucine enhances antimicrobial activity and hemolysis. When changes are done on positions 7 and 10 (on the hydrophilic side) by introducing more positive charges with addition of lysine, both activities also increase. However, when negative charges are introduced instead (with glutamic acids), antimicrobial activity is observed but hemolysis is reduced to zero under the concentrations studied. Although strong inhibitory activity begins at low concentrations (10 μg/mL), some peptides level off inhibition and no change is observed as concentrations are increased.     

A 22-mer Segment in the Structurally Pliable Regulatory Domain of Metazoan CTP: Phosphocholine Cytidylyltransferase Facilitates Both Silencing and Activating Functions

CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, α-synuclein, which we show shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing, we created chimeric enzymes by fusing the catalytic domain of rat CCTα to the regulatory tail of CCTs from Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae or to α-synuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus, both silencing and activating functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or α-synuclein. Deletion of this segment from rat CCT increased the lipid-independent V_max by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus, full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.     

Molecular dissection of the membrane aggregation mechanisms induced by monomeric annexin A2

Annexins are a multigene family of proteins involved in aggregation and fusion processes of biological membranes. One of its best-known members is annexin A2 (or p36), capable of binding to acidic phospholipids in a calcium-dependent manner, as occurs with other members of the same family. In its heterotetrameric form, especially with protein S100A10 (p11), annexin A2 has been involved as a determinant factor in innumerable biological processes like tumor development or anticoagulation. However, the subcellular coexistence of different pools of the protein, in which the monomeric form of annexin A2 is growing in functional relevance, is to date poorly described. In this work we present an exhaustive structural and functional characterization of monomeric human annexin A2 by using different recombinant mutants. The important role of the amphipathic N-terminal α-helix in membrane binding and aggregation has been analyzed. We have also studied the potential implication of lateral “antiparallel” protein dimers in membrane aggregation. In contrast to what was previously suggested, formation of these dimers negatively regulate aggregation. We have also confirmed the essential role of three lysine residues located in the convex surface of the molecule in calcium-free and calcium-dependent membrane binding and aggregation. Finally, we propose models for annexin A2-mediated vesicle aggregation mechanisms.