Characterization of nanodisc-forming peptides for membrane protein studies.

Lipid-bilayer nanodiscs provide a stable, native-like membrane environment for the functional and structural studies of membrane proteins and other membrane-binding molecules. Peptide-based nanodiscs having unique properties are developed for membrane protein studies and other biological applications. While the self-assembly process rendering the formation of peptide-nanodiscs is attractive, it is important to understand the stability and suitability of these nanodisc systems for membrane protein studies. In this study, we investigated the nanodiscs formation by the anti-inflammatory and tumor-suppressing peptide AEM28. AEM28 is a chimeric peptide containing a cationic-rich heparan sulfate proteoglycan- (HSPG)-binding domain from human apolipoprotein E (hapoE) (141-150) followed by the 18A peptide's amino acid sequence. AEM28-based nanodiscs made with different types of lipids were characterized using various biophysical techniques and compared with the nanodiscs formed using 2F or 4F peptides. Variable temperature dynamic light-scattering and 31P NMR experiments indicated the fusion and size heterogeneity of nanodiscs at high temperatures. The suitability of AEM28 and Ac-18A-NH2- (2F-) based nanodiscs for studying membrane proteins is demonstrated by reconstituting and characterizing a drug-metabolizing enzyme, cytochrome-P450 (CYP450), or the redox complex CYP450-CYP450 reductase. AEM28 and 2F were also tested for their efficacies in solubilizing E. coli membranes to understand the possibility of using them for detergent-free membrane protein isolation. Our experimental results suggest that AEM28 nanodiscs are suitable for studying membrane proteins with a net positive charge, whereas 2F-based nanodiscs are compatible with any membrane proteins and their complexes irrespective of their charge. Furthermore, both peptides solubilized E. coli cell membranes, indicating their use in membrane protein isolation and other applications related to membrane solubilization.

Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae: A Structural Analogue of α-Conotoxins.

Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.

A disulfide-stabilised helical hairpin fold in acrorhagin I: An emerging structural motif in peptide toxins.

Acrorhagin I (U-AITX-Aeq5a) is a disulfide-rich peptide identified in the aggressive organs (acrorhagi) of the sea anemone Actinia equina. Previous studies (Toxicon 2005, 46:768-74) found that the peptide is toxic in crabs, although the structural and functional properties of acrorhagin I have not been reported. In this work, an Escherichia coli (BL21 strain) expression system was established for the preparation of 13C,15N-labelled acrorhagin I, and the solution structure was determined using NMR spectroscopy. Structurally, acrorhagin I is similar to B-IV toxin from the marine worm Cerebratulus lacteus (PDB id 1VIB), with a well-defined helical hairpin structure stabilised by four intramolecular disulfide bonds. The recombinant peptide was tested in patch-clamp electrophysiology assays against voltage-gated potassium and sodium channels, and in bacterial and fungal growth inhibitory assays and haemolytic assays. Acrorhagin I was not active against any of the ion channels tested and showed no activity in functional assays, indicating that this peptide may possess a different biological function. Metal ion interaction studies using NMR spectroscopy showed that acrorhagin I bound zinc and nickel, suggesting that its function might be modulated by metal ions or that it may be involved in regulating metal ion levels and their transport. The similarity between the structure of acrorhagin I and that of B-IV toxin from a marine worm suggests that this fold may prove to be a recurring motif in disulfide-rich peptides from marine organisms.

Modulation of Lymphocyte Potassium Channel KV1.3 by Membrane-Penetrating, Joint-Targeting Immunomodulatory Plant Defensin.

We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated KV1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to KV1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for KV1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with 18F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10-100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.

Structural and functional characterisation of a novel peptide from the Australian sea anemone Actinia tenebrosa.

Sea anemone venoms have long been recognised as a rich source of peptides with interesting pharmacological and structural properties. Our recent transcriptomic studies of the Australian sea anemone Actinia tenebrosa have identified a novel 13-residue peptide, U-AITx-Ate1. U-AITx-Ate1 contains a single disulfide bridge and bears no significant homology to previously reported amino acid sequences of peptides from sea anemones or other species. We have produced U-AITx-Ate1 using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The solution structure of U-AITx-Ate1 was determined based on two-dimensional nuclear magnetic resonance spectroscopic data. Diffusion-ordered NMR spectroscopy revealed that U-AITx-Ate1 was monomeric in solution. Perturbations in the 1D 1H NMR spectrum of U-AITx-Ate1 in the presence of dodecylphosphocholine micelles together with molecular dynamics simulations indicated an interaction of U-AITx-Ate1 with lipid membranes, although no binding was detected to 100% POPC and 80% POPC: 20% POPG lipid nanodiscs by isothermal titration calorimetry. Functional assays were performed to explore the biological activity profile of U-AITx-Ate1. U-AITx-Ate1 showed no activity in voltage-clamp electrophysiology assays and no change in behaviour and mortality rates in crustacea. Moderate cytotoxic activity was observed against two breast cancer cell lines.

A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology.

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)-carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline's unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics and NMR experiments. We therefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis.

Identification of the Binding Site of Apical Membrane Antigen†1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

Synthesis, folding, structure and activity of a predicted peptide from the sea anemone Oulactis sp. with an ShKT fold.

Sea anemone venom is rich in bioactive compounds, including peptides containing multiple disulfide bridges. In a transcriptomic study on Oulactis sp., we identified the putative 36-residue peptide, OspTx2b, which is an isoform of the KV channel blocker OspTx2a (Sunanda P et al. [2018] Identification, chemical synthesis, structure and function of a new KV1 channel blocking peptide from Oulactis sp. Peptide Science, in press). As OspTx2b contains a ShK/BgK-like cysteine framework, with high amino acid sequence similarity to BgK, we were interested to investigate its structure and function. The solution structure of OspTx2b was determined using nuclear magnetic resonance spectroscopy. OspTx2b does indeed possess a BgK-like scaffold, with the same disulfide bond connectivities. The orientation of the Lys-Tyr dyad in OspTx2b is more similar to that in ShK than in BgK. However, it failed to show against a range of voltage-gated potassium channels in Xenopus oocytes and human T lymphocytes. OspTx2b also showed no growth inhibitory activity against several strains of bacteria and fungi. Having a BgK-like fold with the Lys-Tyr dyad but no BgK-like activity highlights the importance of key amino acid residues in BgK that are missing in OspTx2b. The lack of activity against the KV channels assessed in this study emphasises that the ShK/BgK scaffold is capable of supporting functional activity beyond potassium channel blockade.

Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK.

Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.

Purification and characterization of adenovirus core protein VII: a histone-like protein that is critical for adenovirus core formation.

Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

The Single Disulfide-Directed ß-Hairpin Fold. Dynamics, Stability, and Engineering.

Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy for enhancing their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed ß-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analogue (rCon-Vc11-22[Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone 15N relaxation measurements for rCon-Vc1[Z1Q] indicate that the N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structure of truncated rCon-Vc11-22[Z1Q] was similar to that of the full-length peptide, indicating that the flexible C-terminus does not have any effect on the structured domain of the peptide. Contryphan-Vc1 exhibited excellent proteolytic stability against trypsin and chymotrypsin but was susceptible to pepsin digestion. We have investigated whether contryphan-Vc1 can accept a bioactive epitope while maintaining the structure of the peptide by introducing peptide sequences based on the DINNN motif of inducible nitric oxide synthase. We show that sCon-Vc11-22[NNN12-14] binds to the iNOS-binding protein SPSB2 with an affinity of 1.3 µM while maintaining the SDH fold. This study serves as a starting point in utilizing the SDH fold as a peptide scaffold.

Theoretical and in vitro studies of a C-terminal peptide from PGKC of Leishmania mexicana mexicana.

Trypanosomatids cause deadly diseases in humans. Of the various biochemical pathways in trypanosomatids, glycolysis, has received special attention because of being sequestered in peroxisome like organelles critical for the survival of the parasites. This study focuses on phosphoglycerate kinase (PGK) from Leishmania spp. which, exists in two isoforms, the cytoplasmic PGKB and glycosomal PGKC differing in their biochemical properties. Computational analysis predicted the likelihood of a transmembrane helix only in the glycosomal isoform PGKC, of approximate length 20 residues in the 62-residue extension, ending at, arginine residues R471 and R472. From experimental studies using circular dichroism and NMR with deuterated sodium dodecyl sulfate, we find that the transmembrane helix spans residues 448±2 to 476 in Leishmania mexicana PGKC. The significance of this observation is discussed in the context of glycosomal transport and substrate tunneling.