Short Α-helical Peptides Research Articles

Antimicrobial peptide 2K4L inhibits the inflammatory response in macrophages and Caenorhabditis elegans and protects against LPS-induced septic shock in mice

2K4L is a rationally designed analog of the short α-helical peptide temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis by substituting amino acid residues. 2K4L displayed improved and broad-spectrum antibacterial activity than temporin-1CEc in vitro. Here, the antibacterial and anti-inflammatory activities of 2K4L in macrophages, C. elegans and mice were investigated. The results demonstrated that 2K4L could enter THP-1 cells to kill a multidrug-resistant Acinetobacter baumannii strain (MRAB 0227) and a sensitive A. baumannii strain (AB 22933), as well as reduce proinflammatory responses induced by MRAB 0227 by inhibiting NF-κB signaling pathway. Similarly, 2K4L exhibited strong bactericidal activity against A. baumannii uptake into C. elegans, extending the lifespan and healthspan of the nematodes. Meanwhile, 2K4L alleviated the oxidative stress response by inhibiting the expression of core genes in the p38 MAPK/PMK-1 signaling pathway and downregulating the phosphorylation level of p38, thereby protecting the nematodes from damage by A. baumannii. Finally, in an LPS-induced septic model, 2K4L enhanced the survival of septic mice and decreased the production of proinflammatory cytokines by inhibiting the signaling protein expression of the MAPK and NF-κB signaling pathways and protecting LPS-induced septic mice from a lethal inflammatory response. In conclusion, 2K4L ameliorated LPS-induced inflammation both in vitro and in vivo.

Unexpected binding modes of inhibitors to the histone chaperone ASF1 revealed by a foldamer scanning approach.

In the search for foldamer inhibitors of the histone chaperone ASF1, we explored the possibility of substituting four α-residues (≈one helix turn) by 3-urea segments and scanned the sequence of a short α-helical peptide known to bind ASF1. By analysing the impact of the different foldamer replacements within the peptide chain, we uncovered new binding modes of the peptide-urea chimeras to ASF1.

NMR “Crystallography” of Membrane Proteins Aligned in Native-Like Bilayers

Oriented-sample (OS) solid-state NMR can be used to determine structures of membrane proteins without crystallization and use of cryogenic temperatures. Here, the orientationally dependent dipolar couplings and chemical shifts provide direct input for structure calculations. However, so far only the 1H-15N dipolar couplings and 15N chemical shifts have been routinely assessed in 15N labeled protein samples. While highly efficient for determining the tilt angles of short α-helical peptides, these two measurements alone are likely insufficient for determining protein structures of arbitrary topology. Therefore, determination of tertiary structures of membrane proteins by OS NMR requires measurements of additional angular restraints. Here we have developed a new experimental triple-resonance NMR technique, which was applied to uniformly doubly (15N, 13C) labeled Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible 1Hα-13Cα dipolar couplings, which represent powerful chiral angular restraints, have been measured for the resolved NMR resonances of the transmembrane domain of Pf1. Inclusion of the third restraint makes it possible to determine the torsion angles Φ and Ψ between the adjacent peptide planes without assuming α-helical structure a priori. Using our recently developed structure calculation algorithm [1], simultaneous fitting of three restraints per peptide plane has resulted in families of structures which have subsequently been filtered using various Rosetta scoring functions including the energetics within the membrane environment, hydrogen bonding, and potential steric clashes. Such structural validation protocol has led to a consensus α-helical transmembrane structure for Pf1 coat protein, albeit obtained in a de novo fashion, thus greatly enhancing utility of OS NMR as a “crystallography” in near-native membrane environments. [1]. Lapin, J. and Nevzorov A.A., Validation of protein backbone structures calculated from NMR angular restraints using Rosetta, J. Biomol. NMR, 2019; 73(5):229-244.

Incorporating Polarizability of Backbone Hydrogen Bonds Improved Folding of Short α-Helical Peptides

Reliability of force fields is an essential aspect of protein-folding simulation. In this work, we introduced a newly developed on-the-fly charge-updating scheme called the polarized structure-specific backbone charge (PSBC) model. The PSBC model was designed with the purpose of building the polarizability of backbone hydrogen bonds into the force field by updating the partial charges of backbone hydrogen-bond donor and acceptor atoms during folding simulation. This implementation was intended to mimic the heterogeneity of the protein surrounding during folding. Multiple single-trajectory molecular dynamics simulations were performed to fold a polyalanine peptide, namely ER (Ac-A(EAAAR)3A-NH2), using both polarizable (PSBC) and nonpolarizable (Amber03) force fields. Through the PSBC model, ER was folded into a helical peptide with helix content that agrees well with experiments. Comparison between simulations performed using the aforementioned force fields demonstrably showed the importance of electrostatic polarization effect in the folding of the short α-helical peptide. The PSBC model was further validated by folding two other short peptides with different helicities.

A powerful ICOS agonist that enhances anti-tumor immune responses restored by immune checkpoint inhibitors

Abstract The Inducible T cell Costimulator (ICOS, CD278) is a receptor in the CD28 family of B7-binding proteins expressed mostly by activated T cells. Upon binding to its ligand ICOS-L expressed on APCs,T cells are further activated by ICOS, resulting in their expansion and production of effector cytokines. Clinical data in patients treated with anti-CTLA-4 or anti-PD-1 mAbs have shown that ICOShiT cells correlated with an increased treatment response in these patients. Here, we report the design of a high avidity pentavalent form of the human ICOS-L extracellular domain fused to a short α-helical peptide from the human cartilage oligomeric matrix protein (COMP). This ICOS agonist, termed ICOS-L.COMP, spontaneously assembles into stable pentamers, binds tightly to both human and murine ICOS (SPR; Kd<10 nM) and co-stimulates (with anti CD3 mAb) the proliferation and cytokine release of both murine and human CD4+ and CD8+ T cells when added as a soluble factor (unlike ICOS-L.Fc). Soluble ICOS-L.COMP also phosphorylates Akt in activated T cells and enhances anti-tumor immune responses in tumor-bearing mice [murine MC38 or CT26 tumors] treated with either anti-murine PD-1 or anti-murine CTLA-4 mAb. Impressively, most mice treated with ICOS-L.COMP combined with an immune checkpoint inhibitor were either cured or displayed a stable tumor burden in contrast to checkpoint monotherapies (delayed tumor progression) or when ICOS-L.COMP was given as a monotherapy (comparable to no treatment). Importantly, ICOS-L.COMP lacks a Fc domain thus eliminating Fc-associated off-target adverse effects. In summary, our results suggest that ICOS-L.COMP represents a new and powerful biologic to be used in combination with existing immune checkpoint inhibitors.

Polyelectrolyte character of rigid rod peptide bundlemer chains constructed via hierarchical self-assembly.

Short α-helical peptides were computationally designed to self-assemble into robust coiled coils that are antiparallel, homotetrameric bundles. These peptide bundle units, or 'bundlemers', have been utilized as anisotropic building blocks to construct bundlemer-based polymers via a hierarchical, hybrid physical-covalent assembly pathway. The bundlemer chains were constructed using short linker connections via 'click' chemistry reactions between the N-termini of bundlemer constituent peptides. The resulting bundlemer chains appear as extremely rigid, cylindrical rods in transmission electron microscopy (TEM) images. Small angle neutron scattering (SANS) shows that these bundlemer chains exist as individual rods in solution with a cross-section that is equal to that of a single coiled coil bundlemer building block of ≈20 Å. SANS further confirms that the interparticle solution structure of the rigid rod bundlemer chains is heterogeneous and responsive to solution conditions, such as ionic-strength and pH. Due to their peptidic constitution, the bundlemer assemblies behave like polyelectrolytes that carry an average charge density of approximately 3 charges per bundlemer as determined from SANS structure factor data fitting, which describes the repulsion between charged rods in solution. This repulsion manifests as a correlation hole in the scattering profile that is suppressed by dilution or addition of salt. Presence of rod cluster aggregates with a mass fractal dimension of ≈2.5 is also confirmed across all samples. The formation of such dense, fractal-like cluster aggregates in a solution of net repulsive rods is a unique example of the subtle balance between short-range attraction and long-rage repulsion interactions in proteins and other biomaterials. With computational control of constituent peptide sequences, it is further possible to deconvolute the underlying sequence driven structure-property relationships in the modular bundlemer chains.

Fabrication of Anti-Icing Surfaces by Short α-Helical Peptides

We designed 12-amino acid peptides as antifreeze protein (AFP) mimetics and tuned the antifreeze activity of the peptides by their structures. Moreover, these short peptides were first immobilized to surfaces as an anti-icing coating. We discovered that the peptides with higher antifreeze activity exhibited better anti-icing performance. It is the first time that short peptides were successfully applied to fabricate anti-icing surfaces, which is certainly advantageous in comparison to the AFP anti-icing coatings previously reported.

Blocking the RecA activity and SOS-response in bacteria with a short α-helical peptide.

The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA filaments at nanomolar concentrations that are two to three orders of magnitude lower than that of RecA protein. Based on the structure of RecX protein complex with the presynaptic RecA filament, we designed a short first in class α-helical peptide that both inhibits RecA protein activities in vitro and blocks the bacterial SOS-response in vivo. The peptide was designed using SEQOPT, a novel method for global sequence optimization of protein α-helices. SEQOPT produces artificial peptide sequences containing only 20 natural amino acids with the maximum possible conformational stability at a given pH, ionic strength, temperature, peptide solubility. It also accounts for restrictions due to known amino acid residues involved in stabilization of protein complexes under consideration. The results indicate that a few key intermolecular interactions inside the RecA protein presynaptic complex are enough to reproduce the main features of the RecX protein mechanism of action. Since the SOS-response provides a major mechanism of bacterial adaptation to antibiotics, these results open new ways for the development of antibiotic co-therapy that would not cause bacterial resistance.

A short D-enantiomeric antimicrobial peptide with potent immunomodulatory and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii

Antimicrobial peptides (AMPs) represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. The present study investigates the antimicrobial activity of new, rationally-designed derivatives of a short α-helical peptide, RR. From the peptides designed, RR4 and its D-enantiomer, D-RR4, emerged as the most potent analogues with a more than 32-fold improvement in antimicrobial activity observed against multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Remarkably, D-RR4 demonstrated potent activity against colistin-resistant strains of P. aeruginosa (isolated from cystic fibrosis patients) indicating a potential therapeutic advantage of this peptide over several AMPs. In contrast to many natural AMPs, D-RR4 retained its activity under challenging physiological conditions (high salts, serum, and acidic pH). Furthermore, D-RR4 was more capable of disrupting P. aeruginosa and A. baumannii biofilms when compared to conventional antibiotics. Of note, D-RR4 was able to bind to lipopolysaccharide to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Finally, D-RR4 protected Caenorhabditis elegans from lethal infections of P. aeruginosa and A. baumannii and enhanced the activity of colistin in vivo against colistin-resistant P. aeruginosa.

Phage Selection of Chemically Stabilized α-Helical Peptide Ligands.

Short α-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development. In recent years, a range of excellent ligands based on stabilized α-helices were generated by rational design using α-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized α-helical ligands in a combinatorial fashion. In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose α-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an α-helical peptide binding β-catenin. We succeeded in developing ligands with Kd's as low as 5.2 nM, having >200-fold improved affinity. The strategy is generally applicable for affinity maturation of any α-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, as no unnatural amino acids are required and the cyclization reaction is more efficient and yields no stereoisomers. A further advantage of the thioether-bridged peptide ligands is that they can be expressed recombinantly as fusion proteins.

A proline-derived transannular N-cap for nucleation of short α-helical peptides.

We report herein a proline-derived transannular N-cap as a helix nucleating template in diverse bio-related peptide sequences via macrolactamization on resin. This approach takes advantage of synergistic stabilization effects of both N-capping properties of proline and substitution of a main chain hydrogen bond with a covalent bond.

Cyclic Peptides as Modulators of Protein-Protein Interactions

Biological processes operate through intricate networks comprised of a myriad of interacting molecules. Protein-protein interactions (PPIs) permeate these networks and are able to transmit and integrate the signalling pathways inside the cells in response to physiological and non-physiological conditions (including cellular pathologies as cancer). To assess the role of such interactions, peptidic compounds have been developed aiming to modulate intracellular PPIs. For instance, structural studies demonstrated in humans and roundworms that the α-helical MAML1 polypeptide binds to a groove formed by the PPI between ICN1 and CLS proteins. The ICN1-CLS-MAML ternary complex plays a key role on NOTCH1 signalling pathway by recruiting the transcription machinery and leading to the transcription of oncogenic NOTCH1 target genes. The group of Dr. James E. Bradner (Harvard Medical School) have designed short α-helical peptides based on a fragment of MAML1 that disrupts the PPI ICN1-CLS, repressing the transcription of NOTCH1 target genes in leukemic cells.

Molecular mechanisms of anticancer action and cell selectivity of short α-helical peptides

Development of functional biomaterials and drugs with good biocompatibility towards host cells but with high potency against cancer cells is a challenging endeavor. By drawing upon the advantageous features of natural antimicrobial peptides and α-helical proteins, we have designed a new class of short α-helical peptides G(IIKK)nI-NH2 (n = 1–4) with different potency and high selectivity against cancer cells. We show that the peptides with n = 3 and 4 kill cancer cells effectively whilst remaining benign to the host cells at their working concentrations, through mechanistic processes similar to their bactericidal effects. The high cell selectivity could stem from their preferential binding to the outer cell membranes containing negative charges and high fluidity. In addition to rapid membrane-permeabilizing capacities, the peptides can also induce the programmed cell death of cancer cells via both mitochondrial pathway and death receptor pathway, without inducing non-specific immunogenic responses. Importantly, these peptides can also inhibit tumor growth in a mouse xenograft model without eliciting side effects. Whilst this study reveals the clinical potential of these peptides as potent drugs and for other medical and healthcare applications, it also points to the significance of fundamental material research in the future development of highly selective peptide functional materials.

Expanding the accessible chemical space by solid phase synthesis of bicyclic homodetic peptides

Norbornapeptides (bicyclo[2.2.1]heptapeptides) and related bicyclic homodetic peptides were prepared by solid-phase peptide synthesis using an orthogonal protection scheme. These conformationally rigid peptides cover an almost pristine area of peptide topological space and adopt globular shapes similar to those of short α-helical peptides.

Ion specific correlations in bulk and at biointerfaces

Ion specific effects are ubiquitous in any complex colloidal or biological fluid inbulk or at interfaces. The molecular origins of these ‘Hofmeister effects’ are notwell understood and their theoretical description poses a formidable challengeto the modeling and simulation community. On the basis of the combinationof atomistically resolved molecular dynamics (MD) computer simulations andstatistical mechanics approaches, we present a few selected examples of specificelectrolyte effects in bulk, at simple neutral and charged interfaces, and on a shortα-helical peptide. The structural complexity in these strongly Coulomb-correlated systems ishighlighted and analyzed in the light of available experimental data. While in general thecomparison of MD simulations to experiments often lacks quantitative agreement, mostlybecause molecular force fields and coarse-graining procedures remain to be optimized, theconsensus as regards trends provides important insights into microscopic hydration andbinding mechanisms.

The role of the central l- or d-Pro residue on structure and mode of action of a cell-selective α-helical IsCT-derived antimicrobial peptide

IsCT-P (ILKKIWKPIKKLF-NH 2) is a novel α-helical antimicrobial peptide with bacterial cell selectivity designed from a scorpion-derived peptide IsCT. To investigate the role of l- or d-Pro kink on the structure and the mode of action of a short α-helical antimicrobial peptide with bacterial cell selectivity, we synthesized IsCT- p, in which d-Pro is substituted for l-Pro 8 of IsCT-P. CD spectra revealed that IsCT-P adopted a typical α-helical structure in various membrane-mimicking conditions, whereas IsCT- p showed a random structure. This result indicated that d-Pro in the central position of a short α-helical peptide provides more remarkable structural flexibility than l-Pro. Despite its higher antibacterial activity, IsCT- p was much less effective at inducing dye leakage in the negatively charged liposome mimicking bacterial membrane and induced no or little membrane potential depolarization of Staphylococcus aureus. Confocal laser scanning microscopy showed that IsCT- p penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas IsCT-P remained outside or on the cell membrane. These results suggested that the major target of IsCT-P and IsCT- p is the bacterial membranes and intracellular components, respectively. Collectively, our results demonstrated that the central d-Pro kink in α-helical antimicrobial peptides plays an important role in penetrating bacterial membrane as well as bacterial cell selectivity.

Stereospecificity of short Rev-derived peptide interactions with RRE IIB RNA.

The essential HIV-1 regulatory protein Rev binds to the Rev responsive element (RRE) of the HIV-1 mRNA. A short alpha-helical peptide derived from Rev (Rev 34-50) and a truncated form of the RRE sequence (RRE IIB) provide a useful in vitro system to study the interactions between Rev and RRE. The current studies focus on evaluating the specificity of the binding interactions between Rev 34-50 and RRE IIB. The binding of L- and D-Rev peptides to natural and enantiomeric RRE IIB RNA was studied by fluorescence spectroscopy. D-Rev and L-Rev peptides bind to RRE IIB with similar affinities. CD measurements are consistent with a nonhelical, probably beta-hairpin, conformation for D-Rev in the complex. The binding affinities of D/L Rev peptides to L-RRE IIB RNA are also similar to those with natural D-RRE IIB. Furthermore, the conformations of L- and D-peptides when bound to L-RRE are reciprocal to the conformations of these peptides in complex with D-RRE. RNA footprinting studies show that L- and D-Rev peptides bind to the same site on RRE IIB. Our results demonstrate lack of stereospecificity in RRE RNA-Rev peptide interactions. However, it is quite possible that the interactions between full-length Rev protein and RRE are highly specific.

A novel method of affinity-purifying proteins using a bis-arsenical fluorescein.

Genetically-encoded affinity tags constitute an important strategy for purifying proteins. Here, we have designed a novel affinity matrix based on the his-arsenical fluorescein dye FlAsH, which specifically recognizes short alpha-helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269-272). We find that kinesin tagged with this cysteine-containing helix binds specifically to FlAsH resin and can be eluted in a fully active form. This affinity tag has several advantages over polyhistidine, the only small affinity tag in common use. The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis tagged kinesin. Moreover, unlike nickel affinity chromatography, which requires high concentrations of imidazole or pH changes for elution, protein bound to the FlAsH column can be completely eluted by dithiothreitol. Because of these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes.

Orientation of α-helical peptides in a lipid bilayer

Samples of pure lipid (dipalmitoylphosphatidylcholine) and lipid containing short α-helical peptides were oriented and examined by X-ray diffraction, together with unoriented samples of pure peptide. X-ray reflections from the bilayer and the α-helices showed that the peptides had oriented in the bilayer with their helical axes perpendicular to the surface.