Biophysical study of the structure and dynamics of the antimicrobial peptide maximin 1.

Maximin 1 is a cationic, amphipathic antimicrobial peptide found in the skin secretions and brains of the Chinese red belly toad Bombina maxima. The 27 amino acid residue-long peptide is biologically interesting as it possesses a variety of biological activities, including antibacterial, antifungal, antiviral, antitumour and spermicidal activities. Its three-dimensional structural model was obtained in a 50/50% water/2,2,2-trifluoroethanol-d3 mixture using two-dimensional NMR spectroscopy. Maximin 1 was found to adopt an α-helical structure from residue Ile2 to Ala26 . The peptide is amphipathic, showing a clear separation between polar and non-polar residues. The interactions with sodium dodecyl sulfate micelles, a widely-used bacterial membrane-mimicking environment, were modelled using molecular dynamics simulations. The peptide maintains an α-helical conformation, occasionally displaying a flexibility around the Gly9 and Gly16 residues, which is likely responsible for the peptide's low haemolytic activity. It is found to preferentially adopt a position parallel to the micellar surface, establishing a number of hydrophobic and electrostatic interactions with the micelle.

Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca.

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defence peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-[Formula: see text] mixture. The structure is defined by an [Formula: see text]-helix that spans between Ile[Formula: see text]-Ala[Formula: see text], and a cyclic disulfide-bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide's interactions with sodium dodecyl sulfate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its [Formula: see text]-helical conformation between residues Ile[Formula: see text]-Ala[Formula: see text], while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

APPTEST is a novel protocol for the automatic prediction of peptide tertiary structures.

Good knowledge of a peptide's tertiary structure is important for understanding its function and its interactions with its biological targets. APPTEST is a novel computational protocol that employs a neural network architecture and simulated annealing methods for the prediction of peptide tertiary structure from the primary sequence. APPTEST works for both linear and cyclic peptides of 5-40 natural amino acids. APPTEST is computationally efficient, returning predicted structures within a number of minutes. APPTEST performance was evaluated on a set of 356 test peptides; the best structure predicted for each peptide deviated by an average of 1.9Å from its experimentally determined backbone conformation, and a native or near-native structure was predicted for 97% of the target sequences. A comparison of APPTEST performance with PEP-FOLD, PEPstrMOD and PepLook across benchmark datasets of short, long and cyclic peptides shows that on average APPTEST produces structures more native than the existing methods in all three categories. This innovative, cutting-edge peptide structure prediction method is available as an online web server at https://research.timmons.eu/apptest, facilitating in silico study and design of peptides by the wider research community.

ENNAVIA is a novel method which employs neural networks for antiviral and anti-coronavirus activity prediction for therapeutic peptides.

Viruses represent one of the greatest threats to human health, necessitating the development of new antiviral drug candidates. Antiviral peptides often possess excellent biological activity and a favourable toxicity profile, and therefore represent a promising field of novel antiviral drugs. As the quantity of sequencing data grows annually, the development of an accurate in silico method for the prediction of peptide antiviral activities is important. This study leverages advances in deep learning and cheminformatics to produce a novel sequence-based deep neural network classifier for the prediction of antiviral peptide activity. The method outperforms the existent best-in-class, with an external test accuracy of 93.9%, Matthews correlation coefficient of 0.87 and an Area Under the Curve of 0.93 on the dataset of experimentally validated peptide activities. This cutting-edge classifier is available as an online web server at https://research.timmons.eu/ennavia, facilitating in silico screening and design of peptide antiviral drugs by the wider research community.

ENNAACT is a novel tool which employs neural networks for anticancer activity classification for therapeutic peptides.

The prevalence of cancer as a threat to human life, responsible for 9.6 million deaths worldwide in 2018, motivates the search for new anticancer agents. While many options are currently available for treatment, these are often expensive and impact the human body unfavourably. Anticancer peptides represent a promising emerging field of anticancer therapeutics, which are characterized by favourable toxicity profile. The development of accurate in silico methods for anticancer peptide prediction is of paramount importance, as the amount of available sequence data is growing each year. This study leverages advances in machine learning research to produce a novel sequence-based deep neural network classifier for anticancer peptide activity. The classifier achieves performance comparable to the best-in-class, with a cross-validated accuracy of 98.3%, Matthews correlation coefficient of 0.91 and an Area Under the Curve of 0.95. This innovative classifier is available as a web server at https://research.timmons.eu/ennaact, facilitating in silico screening and design of new anticancer peptide chemotherapeutics by the research community.

HAPPENN is a novel tool for hemolytic activity prediction for therapeutic peptides which employs neural networks.

The growing prevalence of resistance to antibiotics motivates the search for new antibacterial agents. Antimicrobial peptides are a diverse class of well-studied membrane-active peptides which function as part of the innate host defence system, and form a promising avenue in antibiotic drug research. Some antimicrobial peptides exhibit toxicity against eukaryotic membranes, typically characterised by hemolytic activity assays, but currently, the understanding of what differentiates hemolytic and non-hemolytic peptides is limited. This study leverages advances in machine learning research to produce a novel artificial neural network classifier for the prediction of hemolytic activity from a peptide's primary sequence. The classifier achieves best-in-class performance, with cross-validated accuracy of [Formula: see text] and Matthews correlation coefficient of 0.71. This innovative classifier is available as a web server at https://research.timmons.eu/happenn , allowing the research community to utilise it for in silico screening of peptide drug candidates for high therapeutic efficacies.

Structural and positional studies of the antimicrobial peptide brevinin-1BYa in membrane-mimetic environments.

Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the foothill yellow-legged frog Rana boylii, shows broad-spectrum activity, being particularly effective against opportunistic yeast pathogens. The structure of brevinin-1BYa was investigated in various solution and membrane-mimicking environments by proton nuclear magnetic resonance (1 H-NMR) spectroscopy and molecular modelling. The peptide does not possess a secondary structure in aqueous solution. In a 33% 2,2,2-trifluoroethanol (TFE-d3 )-H2 O solvent mixture, as well as in membrane-mimicking sodium dodecyl sulfate and dodecylphosphocholine micelles, the peptide's structure is characterised by a flexible helix-hinge-helix motif, with the hinge located at the Gly13 /Pro14 residues, and the two α-helices extending from Pro3 to Phe12 and from Pro14 to Thr21 . Positional studies involving the peptide in sodium dodecyl sulfate and dodecylphosphocholine micelles using 5-doxyl-labelled stearic acid and manganese chloride paramagnetic probes show that the peptide's helical segments lie parallel to the micellar surface, with the residues on the hydrophobic face of the amphipathic helices facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that the peptide exerts its biological activity by a non-pore-forming mechanism.

Insights into conformation and membrane interactions of the acyclic and dicarba-bridged brevinin-1BYa antimicrobial peptides.

Brevinin-1BYa is a 24-amino acid residue host-defense peptide, first isolated from skin secretions of the foothill yellow-legged frog Rana boylii. The peptide is of interest, as it shows broad-spectrum antimicrobial activity, and is particularly effective against opportunistic yeast pathogens. Its potential for clinical use, however, is hindered by its latent haemolytic activity. The structures of two analogues, the less haemolytic [C18S,C24S]brevinin-1BYa and the more potent cis-dicarba-brevinin-1BYa, were investigated in various solution and membrane-mimicking environments by [Formula: see text] spectroscopy and molecular modelling techniques. Neither peptide possesses a secondary structure in aqueous solution. In both the membrane-mimicking sodium dodecyl sulphate micelles and 33% 2,2,2-trifluoroethanol ([Formula: see text] solvent mixture, the peptides' structures are characterised by two [Formula: see text]-helices connected by a flexible hinge located at the [Formula: see text] residues. With the aid of molecular dynamics simulations and paramagnetic probes, it was determined that the peptides' helical segments lie parallel to the micellar surface, with their hydrophobic residues facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that both peptides exert their biological activity by a non-pore-forming mechanism. Unlike that of the dicarba analogue, the C-terminus of the acyclic peptide is only weakly associated with the micellar surface and is in direct contact with the surrounding aqueous solvent.

NMR model structure of the antimicrobial peptide maximin 3.

Maximin 3 is a 27-residue-long cationic antimicrobial peptide found in the skin secretion and brain of the Chinese red-belly toad Bombina maxima. The peptide is of biological interest as it possesses anti-HIV activity, not found in the other maximin peptides, in addition to antimicrobial, antitumor and spermicidal activities. The three-dimensional structure of maximin 3 was obtained in a 50/50% water/2,2,2-trifluoroethanol-d3 mixture using two-dimensional NMR spectroscopy. Maximin 3 was found to adopt an α-helical structure from residue G1 to A22, and a coil structure with a helical propensity in the C-terminal tail. The peptide is amphipathic, showing a clear separation between polar and hydrophobic residues. Interactions with sodium dodecyl sulfate micelles, a widely used bacterial membrane-mimicking environment, were modeled using molecular dynamics simulations. The peptide maintained an α-helical conformation, occasionally displaying a flexibility around residues G9 and G16, which is likely responsible for the peptide's low haemolytic activity. It is found to preferentially adopt a position parallel to the micellar surface, establishing a number of hydrophobic and electrostatic interactions with it.

A 13C-NMR study of azacryptand complexes.

An azacryptand has been solubilised in aqueous media containing 50% (v/v) dimethyl sulphoxide. (13)C-NMR has been used to determine how the azacryptand is affected by zinc binding at pH 10. Using (13)C-NMR and (13)C-enriched bicarbonate we have been able to observe the formation of 4 different carbamate derivatives of the azacryptand at pH 10. The azacryptand was shown to solubilise zinc or cadmium at alkaline pHs. Two moles of zinc are bound per mole of azacryptand and this complex binds 1 mole of carbonate. By replacing the zinc with cadmium-113 we have shown that the (13)C-NMR signal of the (13)C-enriched carbon of the bound carbonate is split into two triplets at 2.2 °C. This shows that two cadmium complexes are formed and in each of these complexes the carbonate group is bound by two magnetically equivalent metal ions. It also demonstrates that these cadmium complexes are not in fast exchange. From temperature studies we show that in the zinc complexes both complexes are in fast exchange with each other but are in slow exchange with free bicarbonate. HOESY is used to determine the position of the carbonate carbon in the complex. The solution and crystal structures of the zinc-carbonate-azacryptand complexes are compared.

Structural aspects of gut peptides with therapeutic potential for type 2 diabetes.

Gut hormones represent a niche subset of pharmacologically active agents that are rapidly gaining importance in medicine. Due to their exceptional specificity for their receptors, these hormones along with their analogues have attracted considerable pharmaceutical interest for the treatment of human disorders including type 2 diabetes. With the recent advances in the structural biology, a significant amount of structural information for these hormones is now available. This Minireview presents an overview of the structural aspects of these hormones, which have roles in physiological processes such as insulin secretion, as well as a discussion on the relevant structural modifications used to improve these hormones for the treatment of type 2 diabetes.

Racemisation of N-Fmoc phenylglycine under mild microwave-SPPS and conventional stepwise SPPS conditions: attempts to develop strategies for overcoming this.

We have been engaged in the microwave-solid phase peptide synthesis (SPPS) synthesis of the phenylglycine (Phg)-containing pentapeptide H-Ala-Val-Pro-Phg-Tyr-NH(2) (1) previously demonstrated to bind to the so-called BIR3 domain of the anti-apoptotic protein XIAP. Analysis of the target peptide by a combination of RP-HPLC, ESI-MS, and NMR revealed the presence of two diastereoisomers arising out of the racemisation of the Phg residue, with the percentage of the LLLDL component assessed as 49%. We performed the synthesis of peptide (1) using different microwave and conventional stepwise SPPS conditions in attempts to reduce the level of racemisation of the Phg residue and to determine at which part of the synthetic cycle the epimerization had occurred. We determined that racemisation occurred mainly during the Fmoc-group removal and, to a much lesser extent, during activation/coupling of the Fmoc-Phg-OH residue. We were able to obtain the desired peptide with a 71% diastereomeric purity (29% LLLDL as impurity) by utilizing microwave-assisted SPPS at 50 °C and power 22 Watts, when the triazine-derived coupling reagent DMTMM-BF(4) was used, together with NMM as an activator base, for the incorporation of this residue and 20% piperidine as an Fmoc-deprotection base. In contrast, the phenylalanine analogue of the above peptide, H-Ala-Val-Pro-Phe-Tyr-NH(2) (2), was always obtained as a single diastereoisomer by using a range of standard coupling and deprotection conditions. Our findings suggest that the racemisation of Fmoc-Phg-OH, under both microwave-SPPS and stepwise conventional SPPS syntheses conditions, is very facile but can be limited through the use of the above stated conditions.

Conformational and membrane interaction studies of the antimicrobial peptide alyteserin-1c and its analogue [E4K]alyteserin-1c.

Alyteserin-1c (GLKEIFKAGLGSLVKGIAAHVAS.NH(2)), first isolated from skin secretions of the midwife toad Alytes obstetricans, shows selective growth-inhibitory activity against Gram-negative bacteria. The structures of alyteserin-1c and its more potent and less haemolytic analogue [E4K]alyteserin-1c were investigated in various solution and membrane mimicking environments by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide displays a lack of secondary structure but, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure is characterised by an extended alpha helix between residues Leu(2) and Val(21). Solution structural studies in the membrane mimicking environments, sodium dodecyl sulphate (SDS), dodecylphosphocholine (DPC), and 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) micelles, indicate that these peptides display an alpha helical structure between residues Lys(3) and Val(21). Positional studies of the peptides in SDS, DPC and DHPC media show that the N-terminal and central residues lie inside the micelle while C-terminal residues beyond Ala(19) do not interact with the micelles.

Conformational, receptor interaction and alanine scan studies of glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP) is an insulinotropic incretin hormone that stimulates insulin secretion during a meal. GIP has glucose lowering abilities and hence is considered as a potential target molecule for type 2 diabetes therapy. In this article, we present the solution structure of GIP in membrane-mimicking environments by proton NMR spectroscopy and molecular modelling. GIP adopts an α-helical conformation between residues Phe(6)-Gly(31) and Ala(13)-Gln(29) for micellar and bicellar media, respectively. Previously we examined the effect of N-terminal Ala substitution in GIP, but here eight GIP analogues were synthesised by replacing individual residues within the central 8-18 region with alanine. These studies showed relatively minor changes in biological activity as assessed by insulin releasing potency. However, at higher concentration, GIP(Ala(16)), and GIP(Ala(18)) showed insulin secreting activity higher than the native GIP (P<0.01 to P<0.001) in cultured pancreatic BRIN-BD11 cells. Receptor interaction studies of the native GIP with the extracellular domain of its receptor were performed by using two different docking algorithms. At the optimised docking conformation, the complex was stabilised by the presence of hydrophobic interactions and intermolecular hydrogen bonding. Further, we have identified some potentially important additional C-terminal interactions of GIP with its N-terminal extracellular receptor domain.

Remeasuring HEWL pK(a) values by NMR spectroscopy: methods, analysis, accuracy, and implications for theoretical pK(a) calculations.

Site-specific pK(a) values measured by NMR spectroscopy provide essential information on protein electrostatics, the pH-dependence of protein structure, dynamics and function, and constitute an important benchmark for protein pK(a) calculation algorithms. Titration curves can be measured by tracking the NMR chemical shifts of several reporter nuclei versus sample pH. However, careful analysis of these curves is needed to extract residue-specific pK(a) values since pH-dependent chemical shift changes can arise from many sources, including through-bond inductive effects, through-space electric field effects, and conformational changes. We have re-measured titration curves for all carboxylates and His 15 in Hen Egg White Lysozyme (HEWL) by recording the pH-dependent chemical shifts of all backbone amide nitrogens and protons, Asp/Glu side chain protons and carboxyl carbons, and imidazole protonated carbons and protons in this protein. We extracted pK(a) values from the resulting titration curves using standard fitting methods, and compared these values to each other, and with those measured previously by ¹H NMR (Bartik et al., Biophys J 1994;66:1180­1184). This analysis gives insights into the true accuracy associated with experimentally measured pK(a) values. We find that apparent pK(a) values frequently differ by 0.5­1.0 units depending upon the nuclei monitored, and that larger differences occasionally can be observed. The variation in measured pK(a) values, which reflects the difficulty in fitting and assigning pH-dependent chemical shifts to specific ionization equilibria, has significant implications for the experimental procedures used for measuring protein pK(a) values, for the benchmarking of protein pK(a) calculation algorithms, and for the understanding of protein electrostatics in general.

In vitro activities of synthetic host defense propeptides processed by neutrophil elastase against cystic fibrosis pathogens.

The antimicrobial and hemolytic activities of a host defense peptide can be controlled by its modification as a propeptide of reduced net charge, which can then be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis.

Conformational and molecular interaction studies of glucagon-like peptide-2 with its N-terminal extracellular receptor domain.

Glucagon-like peptide-2 (GLP-2) is a therapeutic target used in the treatment of short bowel syndrome. In this paper, we present the three dimensional solution structure of GLP-2 peptide determined using nuclear magnetic resonance (NMR) and molecular modelling. The GLP-2 adopts an α-helical conformation similar to that of secretin family of hormones. In order to understand the molecular details governing the ligand binding and receptor activation, macromolecular docking studies were performed between the N-terminal extracellular domain of GLP-2 receptor and the GLP-2 hormone using a data driven docking program.

Metabolic and structural properties of human obestatin {1-23} and two fragment peptides.

Obestatin is a peptide produced in the oxyntic mucosa of the stomach and co-localizes with ghrelin on the periphery of pancreatic islets. Several studies demonstrate that obestatin reduces food and water intake, decreases body weight gain, inhibits gastrointestinal motility, and modulates glucose-induced insulin secretion. In this study we evaluated the acute metabolic effects of human obestatin {1-23} and fragment peptides {1-10} or {11-23} in high-fat fed mice, and then investigated their solution structure by NMR spectroscopy and molecular modelling. Obestatins {1-23} and {11-23} significantly reduced food intake (86% and 90% respectively) and lowered glucose responses to feeding, whilst leaving insulin responses unchanged. No metabolic changes could be detected following the administration of obestatin {1-10}. In aqueous solution none of the obestatin peptides possessed secondary structural features. However, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure of obestatin {1-23} was characterized by an alpha-helix followed by a single turn helix conformation between residues Pro(4) and Gln(15) and His(19) and Ala(22) respectively. Obestatin {1-10} showed no structural components whereas {11-23} contained an alpha-helix between residues Val(14) and Ser(20) in a mixed solvent. These studies are the first to elucidate the structure of human obestatin and provide clear evidence that the observed alpha-helical structures are critical for in vivo activity. Future structure/function studies may facilitate the design of novel therapeutic agents based on the obestatin peptide structure.

Development of potent anti-infective agents from Silurana tropicalis: conformational analysis of the amphipathic, alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7.

Peptide XT-7 (GLLGP(5)LLKIA(10)AKVGS(15)NLL.NH(2)) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC(50)=140 microM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC(50)>500 microM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O mixed solvent system, XT-7 is characterised by a right handed alpha-helical conformation between residues Leu(3) and Leu(17) whereas [G4K]XT-7 adopts a more restricted alpha-helical conformation between residues Leu(6) and Leu(17). A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu(3) and Leu(17). However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H(2)O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly(4)-->Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.

Identification of determinants of glucose-dependent insulinotropic polypeptide receptor that interact with N-terminal biologically active region of the natural ligand.

Glucose-dependent insulinotropic polypeptide receptor (GIPR), a member of family B of the G-protein coupled receptors, is a potential therapeutic target for which discovery of nonpeptide ligands is highly desirable. Structure-activity relationship studies indicated that the N-terminal part of glucose-dependent insulinotropic polypeptide (GIP) is crucial for biological activity. Here, we aimed at identification of residues in the GIPR involved in functional interaction with N-terminal moiety of GIP. A homology model of the transmembrane core of GIPR was constructed, whereas a three-dimensional model of the complex formed between GIP and the N-terminal extracellular domain of GIPR was taken from the crystal structure. The latter complex was docked to the transmembrane domains of GIPR, allowing in silico identification of putative residues of the agonist binding/activation site. All mutants were expressed at the surface of human embryonic kidney 293 cells as indicated by flow cytometry and confocal microscopy analysis of fluorescent GIP binding. Mutation of residues Arg183, Arg190, Arg300, and Phe357 caused shifts of 76-, 71-, 42-, and 16-fold in the potency to induce cAMP formation, respectively. Further characterization of these mutants, including tests with alanine-substituted GIP analogs, were in agreement with interaction of Glu3 in GIP with Arg183 in GIPR. Furthermore, they strongly supported a binding mode of GIP to GIPR in which the N-terminal moiety of GIP was sited within transmembrane helices (TMH) 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5), and Phe357 (TMH6). These data represent an important step toward understanding activation of GIPR by GIP, which should facilitate the rational design of therapeutic agents.