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Peptides with a combination of high positive charge and high hydrophobicity have high antimicrobial activity, as epitomized by peptide venoms, which are designed by nature as disruptors of host membranes yet also display significant efficacy against pathogens. To investigate this phenomenon systematically, here we focus on ponericin W1, a peptide venom isolated from Pachycondyla goeldii ants (WLGSALKIGAKLLPSVVGLFKKKKQ) to examine whether Lys positioning can be broadly applied to optimize the functional range of existing natural sequences. We prepared sets of ponericin W1 analogues, where Lys residues were either distributed in an amphipathic manner throughout the sequence (PonAmp), clustered at the N-terminus (PonN), or clustered at the C-terminus (PonC), along with their counterparts of reduced hydrophobicity through 2-4 Leu-to-Ala replacements. We found that wild-type ponericin W1 and all three variants displayed toxicity against human erythrocytes, but hemolysis was eliminated by the replacement of two or more Leu residues by Ala residues. As well, peptides containing up to 3 Leu-to-Ala replacements retained antimicrobial activity against E. coli bacteria. Biophysical analyses of peptide-membrane interaction patterns by circular dichroism spectroscopy revealed a novel mode of cluster-dependent peptide positioning vis-a-vis the water-membrane interface, where PonAmp and PonC peptides displayed full or partial helical structures, while PonN peptides were unstructured, likely due, in part, to dynamic interchange between aqueous and membrane surface environments. The overall findings suggest that the lower membrane penetration of N-terminal charge-clustered constructs coupled with moderate sequence hydrophobicity may be advantageous for conferring enhanced target selectivity for bacterial versus mammalian membranes.

Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the drugs can exert their intended toxic effect. The Escherichia coli membrane protein AcrB is the archetypal protein utilized for bacterial efflux study because it can extrude a diverse range of antibiotic substrates and has close homologues in many Gram-negative pathogens. Three AcrB subunits, each of which contains 12 transmembrane (TM) helices, are known to trimerize to form the minimal functional unit, stabilized noncovalently by helix-helix interactions between TM1 and TM8. To inhibit the efflux activity of AcrB, we have rationally designed synthetic peptides aimed at destabilizing the AcrB trimerization interface by outcompeting the subunit interaction sites within the membrane. Here we report that peptides mimicking TM1 or TM8, with flanking N-terminal peptoid tags, and C-terminal lysine tags that aid in directing the peptides to their membrane-embedded target, decrease the AcrB-mediated efflux of the fluorescent substrate Nile red and potentiate the effect of the antimicrobials chloramphenicol and ethidium bromide. To further characterize the motif encompassing the interaction between TM1 and TM8, we used Forster resonance energy transfer to demonstrate dimerization. Using the TM1 and TM8 peptides, in conjunction with several selected mutant peptides, we highlight residues that may increase the potency and specificity of the peptide drug candidates. In targeting membrane-embedded protein-protein interactions, this work represents a novel approach to AcrB inhibition and, more broadly, a potential route to a new category of efflux pump inhibitors.

Chronic infection and inflammation are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. ORKAMBI&reg, (Lumacaftor-Ivacaftor) is an approved combination therapy for Cystic Fibrosis (CF) patients bearing the most common mutation, F508del, in the cystic fibrosis conductance regulator (CFTR) protein. It has been previously shown that ORKAMBI&reg, mediated rescue of CFTR is reduced by a pre-existing Pseudomonas aeruginosa infection. Here, we show that the infection of F508del-CFTR human bronchial epithelial (HBE) cells with lab strain and four different clinical strains of P. aeruginosa, isolated from the lung sputum of CF patients, decreases CFTR function in a strain-specific manner by 48 to 88%. The treatment of infected cells with antibiotic tobramycin or cationic antimicrobial peptide 6K-F17 was found to decrease clinical strain bacterial growth on HBE cells and restore ORKAMBI&reg, mediated rescue of F508del-CFTR function. Further, 6K-F17 was found to downregulate the expression of pro-inflammatory cytokines, interleukin (IL)-8, IL-6, and tumor necrosis factor-&alpha, in infected HBE cells. The results provide strong evidence for a combination therapy approach involving CFTR modulators and anti-infectives (i.e., tobramycin and/or 6K-F17) to improve their overall efficacy in CF patients.

Our meagre understanding of CFTR misfolding and its reversal by small-molecule correctors hampers the development of mechanism-based therapies of cystic fibrosis. Here we exploit a helical-hairpin construct—the simplest proxy of membrane-protein tertiary contacts—containing CFTR’s transmembrane helices 3 and 4 and its corresponding disease phenotypic mutant V232D to gain molecular-level insights into CFTR misfolding and drug rescue by the corrector Lumacaftor. Using a single-molecule FRET approach to study hairpin conformations in lipid bilayers, we find that the wild-type hairpin is well folded, whereas the V232D mutant assumes an open conformation in bilayer thicknesses mimicking the endoplasmic reticulum. Addition of Lumacaftor reverses the aberrant opening of the mutant hairpin to restore a compact state as in the wild type. The observed membrane escape of the V232D hairpin and its reversal by Lumacaftor complement cell-based analyses of the full-length protein, thereby providing in vivo and in vitro correlates of CFTR misfolding and drug-action mechanisms., Georg Krainer and Antoine Treff et al. use a helical-hairpin construct derived from the cystic fibrosis transmembrane conductance regulator (CFTR) to investigate misfolding caused by the disease-linked V232D mutation. Using single-molecule FRET, they show that the V232D hairpin assumes an open conformation in lipid bilayers, which is reversed by the pharmacological corrector Lumacaftor.

Cyclization has been recognized as a valuable technique for increasing the efficacy of small molecule and peptide therapeutics. Here we report the application of a hydrocarbon staple to a rationally-designed cationic antimicrobial peptide (CAP) that acquires increased membrane targeting and interaction vs. its linear counterpart. The previously-described CAP, 6K-F17 (KKKKKK-AAFAAWAAFAA-NH2) was used as the backbone for incorporation of an i to i + 4 helical hydrocarbon staple through olefin ring closing metathesis. Stapled versions of 6K-F17 showed an increase in non-selective membrane interaction, where the staple itself enhances the degree of membrane interaction and rate of cell death while maintaining high potency against bacterial membranes. However, the higher averaged hydrophobicity imparted by the staple also significantly increases toxicity to mammalian cells. This deleterious effect is countered through stepwise reduction of the stapled 6K-F17’s backbone hydrophobicity through polar amino acid substitutions. Circular dichroism assessment of secondary structure in various bacterial membrane mimetics reveals that a helical structure may improve – but is not an absolute requirement for – antimicrobial activity of 6K-F17. Further, phosphorus-31 static solid state NMR spectra revealed that both non-toxic stapled and linear peptides bind bacterial membranes in a similar manner that does not involve a detergent-like mechanism of lipid removal. The overall results suggest that the technique of hydrocarbon stapling can be readily applied to membrane-interactive CAPs to modulate how they interact and target biological membranes.

Significance Using D-amino acids as the building blocks for bioactive peptides can dramatically increase their potency. However, simply swapping regular levorotary amino acids for dextrorotary (D)-amino acids alters the peptide surface topology and function is lost. Current methods to overcome this are not generally applicable and exclude the majority of therapeutic targets. By creating a mirror image of all 111,867 protein structures in the Protein Data Bank (PDB), we convert this repository into a D-peptide database with 2.8 million D-peptide structures. This D-PDB can be searched to find therapeutically active topologies, demonstrated here by the discovery of D-peptide GLP1R and PTH1R agonists. Evaluation of D-PDB coverage suggests that it holds candidates for most therapeutic targets and, thus, potentially contains hundreds of potent drug leads., Biologics are a rapidly growing class of therapeutics with many advantages over traditional small molecule drugs. A major obstacle to their development is that proteins and peptides are easily destroyed by proteases and, thus, typically have prohibitively short half-lives in human gut, plasma, and cells. One of the most effective ways to prevent degradation is to engineer analogs from dextrorotary (D)-amino acids, with up to 105-fold improvements in potency reported. We here propose a general peptide-engineering platform that overcomes limitations of previous methods. By creating a mirror image of every structure in the Protein Data Bank (PDB), we generate a database of ∼2.8 million D-peptides. To obtain a D-analog of a given peptide, we search the (D)-PDB for similar configurations of its critical—“hotspot”—residues. As a proof of concept, we apply our method to two peptides that are Food and Drug Administration approved as therapeutics for diabetes and osteoporosis, respectively. We obtain D-analogs that activate the GLP1 and PTH1 receptors with the same efficacy as their natural counterparts and show greatly increased half-life.

Membrane proteins play the central roles in a variety of cellular processes, ranging from nutrient uptake and signalling, to cell-cell communication. Their biological functions are directly related to how they fold and assemble; defects often lead to disease. Protein-protein interactions (PPIs) within the membrane are therefore of great interest as therapeutic targets. Here we review the progress in the application of membrane-insertable peptides for the disruption or stabilization of membrane-based PPIs. We describe the design and preparation of transmembrane peptide mimics; and of several categories of peptidomimetics used for study, including d-enantiomers, non-natural amino acids, peptoids, and β-peptides. Further aspects of the review describe modifications to membrane-insertable peptides, including lipidation and cyclization via hydrocarbon stapling. These approaches provide a pathway toward the development of metabolically stable, non-toxic, and efficacious peptide modulators of membrane-based PPIs. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Bacteria have acquired multiple mechanisms to evade the lethal effects of current therapeutics, hindering treatment of bacterial infections, such as those caused by the pathogen Pseudomonas aeruginosa, which is responsible for nosocomial and cystic fibrosis lung infections. One resistance mechanism involves membrane-embedded multidrug efflux pumps that can effectively extrude an array of substrates, including common antibiotics, dyes, and biocides. Among these is a small multidrug resistance (SMR) efflux protein, consisting of four transmembrane (TM) helices, that functions as an antiparallel dimer. TM helices 1 to 3 (TM1 to TM3) comprise the substrate binding pocket, while TM4 contains a GG7 heptad sequence motif that mediates the SMR TM4-TM4 dimerization. In the present work, we synthesized a series of peptides containing the residues centered on the TM4-TM4 binding interface found in the P. aeruginosa SMR (PAsmr), typified by Ac-Ala-(Sar)3-LL GI G LI IA GV LV-KKK-NH2 (helix-helix interaction residues are underlined). Here, the acetylated N-terminal sarcosine (N-methyl-Gly) tag [Ac-Ala-(Sar)3] promotes membrane penetration, while the C-terminal Lys tag promotes selectivity for the negatively charged bacterial membranes. This peptide was observed to competitively disrupt PAsmr-mediated efflux, as measured by efflux inhibition of the fluorescent dye ethidium bromide, while having no effect on cell membrane integrity. Alternatively, a corresponding peptide in which the TM4 binding motif is scrambled was inactive in this assay. In addition, when Escherichia coli cells expressing PAsmr were combined with sublethal concentrations of several biocides, growth was significantly inhibited when peptide was added, notably, by up to 95% with the disinfectant benzylalkonium chloride. These results demonstrate promise for an efflux pump inhibitor to address the increasing threat of antibiotic-resistant bacteria.

Natural α-helical cationic antimicrobial peptide (CAP) sequences are predominantly amphipathic, with only ca. 2% containing four or more consecutive positively charged amino acids (Lys/Arg). We have designed synthetic CAPs that deviate from these natural sequences, as typified by the charge-clustered peptide KKKKKKAAFAAWAAFAA-NH2, (termed 6K-F17), which displays high antimicrobial activity with no toxicity to mammalian cells. We created a series of peptides varying in charge patterning, increasing the amphipathic character of 6K-F17 to mimic the design of natural CAPs (e.g., KAAKKFAKAWAKAFAA-NH2). Amphipathic sequences displayed increased antimicrobial activity against bacteria but were significantly more toxic to mammalian cells and more susceptible to protease degradation than their corresponding charge-clustered variants, suggesting that amphipathic sequences may be desirable in nature to allow for more versatile functions (i.e., antibacterial, antifungal, antipredator) and rapid clearance from vulnerable host cells. Our approach to clustering of charges may therefore allow for specialization against bacteria, in concert with prolonged peptide half-life.

Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is complicated by the diverse environments (aqueous vs lipidic) the protein encounters and by the fact that most TM segments contain a substantial amount (∼30%) of polar residues, as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical, but sequentially different, peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous "block" in the middle of the segment or "scrambled" throughout the sequence) and (ii) in Ser content (0-6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima, as well as by the extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum. We found that local blocks of high hydrophobicity heighten the translocon's propensity to insert moderately hydrophilic sequences, until a "threshold hydrophilicity" is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomalous translocation/insertion.

With the increasing recognition of biofilms in human disease, the development of novel antimicrobial therapies is of critical importance. For example, in patients with cystic fibrosis (CF), the acquisition of host-adapted, chronic Pseudomonas aeruginosa infection is associated with a decline in lung function and increased mortality. Our objective was to test the in vitro efficacy of a membrane-active antimicrobial peptide we designed, termed 6K-F17 (sequence: KKKKKK-AAFAAWAAFAA-NH2), against multidrug resistant P. aeruginosa biofilms. This peptide displays high antimicrobial activity against a range of pathogenic bacteria, yet is non-hemolytic to human erythrocytes and non-toxic to human bronchial epithelial cells. In the present work, P. aeruginosa strain PAO1, and four multidrug resistant (MDR) isolates from chronically infected CF individuals, were grown as 48-hour biofilms in a static biofilm slide chamber model. These biofilms were then exposed to varying concentrations of 6K-F17 alone, or in the presence of tobramycin, prior to confocal imaging. Biofilm biovolume and viability were assessed. 6K-F17 was able to kill biofilms – even in the presence of sputum – and greatly reduce biofilm biovolume in PAO1 and MDR isolates. Strikingly, when used in conjunction with tobramycin, low doses of 6K-F17 significantly potentiated tobramycin killing, leading to biofilm destruction.

Small multidrug resistance (SMR) pumps represent a minimal paradigm of proton-coupled membrane transport in bacteria, yet no high-resolution structure of an SMR protein is available. Here, atomic-resolution structures of the Escherichia coli efflux-multidrug resistance E (EmrE) multidrug transporter in ligand-bound form are refined using microsecond molecular dynamics simulations biased using low-resolution data from X-ray crystallography. The structures are compatible with existing mutagenesis data as well as NMR and biochemical experiments, including pKas of the catalytic glutamate residues and the dissociation constant ([Formula: see text]) of the tetraphenylphosphonium(+) cation. The refined structures show the arrangement of residue side chains in the EmrE active site occupied by two different ligands and in the absence of a ligand, illustrating how EmrE can adopt structurally diverse active site configurations. The structures also show a stable, well-packed binding interface between the helices H4 of the two monomers, which is believed to be crucial for EmrE dimerization. Guided by the atomic details of this interface, we design proteolysis-resistant stapled peptides that bind to helix H4 of an EmrE monomer. The peptides are expected to interfere with the dimerization and thereby inhibit drug transport. Optimal positions of the peptide staple were determined using free-energy simulations of peptide binding to monomeric EmrE. Three of the four top-scoring peptides selected for experimental testing resulted in significant inhibition of proton-driven ethidium efflux in live cells without nonspecific toxicity. The approach described here is expected to be of general use for the design of peptide therapeutics.

Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in Leu-block segments, promote translocon-mediated insertion.

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations - glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.

Cystic fibrosis (CF) is the most common lethal genetic disease among the Western population caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The development of effective therapeutic agents against CF requires a molecular understanding of how mutation-related structural alterations lead to impaired functioning of CFTR. The main cause for CF are processing mutations frequently found within CFTR-transmembrane (TM) segments which affect co-translational folding and insertion. However, the underlying molecular mechanisms for misfolding remain obscure, mainly because of the unavailability of high-resolution structures and a lack of methods suitable for studying membrane protein structure-function relationships.Devising a divide-and-conquer approach in combination with single-molecule FRET (smFRET), we present here a strategy to gain novel insights into the structural basis for conformational misfolding in CFTR. Using this approach, we study the CF-phenotypic polar mutation V232D located within the fourth TM helix (TM4) of CFTR. In vivo folding and insertion of TM4 is functionally coupled with TM3 where both TM segments are co-translationally inserted into the membrane as a TM helix-loop-helix hairpin motif (TM3/4). Utilizing this minimal folding unit, we employ smFRET as a molecular ruler to readout structural alterations imposed on the helical packing of the TM3/4 hairpin upon mutation. In contrast to earlier findings that suggested a TM lock between TM3 and TM4 by a non-native H-bond, our results reveal that V232D TM3/4 associates with membranes in an open conformation. This implies that the charged residue imposes an energy penalty on membrane insertion of the hairpin. We propose a model for CF pathogenesis where partitioning of V232D TM3/4 into the interfacial region leads to misfolding of CFTR during co-translational membrane insertion and inhibits maturation by trapping the protein as a partially folded intermediate.

Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues (90)GLALIVAGV(98)); and (ii) a peptide comprised of the full TM4(85-105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88-100), and then "staple" this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-(88)VVGLXLIZXGVVV(100)-KKK-NH2 (X = 2-(4'-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn(95) peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88-100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes.