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Context: BIM (Bcl-2 interacting mediator of apoptosis)-derived peptides that specifically target over-expressed Mcl-1 (myeloid cell leukemia-1) protein and induce apoptosis are potentially anti-cancer agents. Since the helicity of BIM-derived peptides has a crucial role in their functionality, a range of strategies have been used to increase the helicity including the introduction of unnatural residues and stapling methods that have some drawbacks such as the accumulation in the liver. To avoid these drawbacks, this study aimed to design a more helical peptide by utilizing bioinformatics algorithms and molecular dynamics simulations without exploiting unnatural residues and stapling methods. MM-PBSA results showed that the mutations of A4fE and A2eE in analogue 5 demonstrate a preference towards binding with Mcl-1. As evidenced by Circular dichroism results, the helicity increases from 18 to 34%, these findings could enhance the potential of analogue 5 as an anti-cancer agent targeting Mcl-1. The applied strategies in this research could shed light on the in silico peptide design. Moreover, analogue 5 as a drug candidate can be evaluated in vitro and in vivo studies. Methods: The sequence of the lead peptide was determined using the ApInAPDB database and PRALINE program. Contact finder and PDBsum web server softwares were used to determine the contact involved amino acids in complex with Mcl-1. All identified salt bridge contributing residues were unaltered to preserve the binding affinity. After proposing novel analogues, their secondary structures were predicted by Cham finder web server software and GOR, Neural Network, and Chou-Fasman algorithms. Finally, molecular dynamics simulations run for 100 ns were done using the GROMACS, version 5.0.7, with the CHARMM36 force field. MM-PBSA was used to assess binding affinity specificity in targeting Mcl-1 and Bcl-xL (B-cell lymphoma extra-large). [ABSTRACT FROM AUTHOR]

Cyclic peptides are widespread throughout the plant kingdom, and display diverse sequences, structures and bioactivities. The potential applications attributed to these peptides and their unusual biosynthesis has captivated the attention of researchers for many years. Several gene sequences for plant cyclic peptides have been discovered over the last two decades but it is only recently that we are beginning to understand the intricacies associated with their biosynthesis. Recent studies have focussed on three main classes of plant derived cyclic peptides, namely orbitides, SFTI related peptides and cyclotides. In this mini-review, we discuss the expansion of the known sequence and structural diversity in these families, insights into the enzymes involved in the biosynthesis, the exciting applications which includes a cyclotide currently in clinical trials for the treatment of multiple sclerosis, and new production methods that are being developed to realise the potential of plant cyclic peptides as pharmaceutical or agricultural agents. [ABSTRACT FROM AUTHOR]

Abstract Arthropods are a diverse and ancient group of invertebrate animals, which constitute approximately 75–85% of all known species on earth. Many arthropod species, such as spiders, scorpions and even some crustaceans, contain venoms that can be very complex, representing natural combinatorial libraries of bioactive compounds. Characterization of the compounds in these libraries has helped the development of tools for pharmacology, and the discovery of lead molecules for therapeutic treatments. A critical aspect in the characterization of venom compounds is determination of three-dimensional structure. Structural analysis can provide insight into many processes including understanding target interactions and developing more potent and selective drug leads. Arthropod venoms are an extremely rich source of novel structures and this review provides an overview of this structural diversity, including structures of proteins, peptides and small molecules. Highlights • Arthropod venoms have enormous structural diversity, and include proteins, peptides and small molecules. • Peptides are highly prevalent in these venoms with the inhibitor cystine knot structural motif well represented. • Recent studies have highlighted different classes of ICK peptides and provided insight into the evolution of this motif. • Structural studies have helped in elucidating the function of these arthropod venom components. [ABSTRACT FROM AUTHOR]

Over the last two decades, the cyclotides have generated widespread interest both in the plant biology and peptide communities for a number of reasons, including their physiological roles in plants, evolution, and wide range of biological activities with potential therapeutic applications. But perhaps most of all it is their unique structure with a cyclic cystine knot motif that provide remarkable features such as resistance to chemical and biological degradation, which have caught researchers' attention. In this chapter, we summarize the key studies that have contributed to our understanding of the cyclotide fold, the structural features of these peptides, and the role and utilization of these features for native and engineered biological functions. [ABSTRACT FROM AUTHOR]

In the past 100 years minimal venom research has focused on ticks despite several species possessing a highly paralytic and lethal venom cocktail of proteinaceous molecules. The saliva of the Australian paralysis tick, Ixodes holocyclus , has been responsible for 20 human fatalities from 1900 to 1945, and up to 100,000 domestic animal fatalities annually. In the last 50 years, research on this tick has focused on identifying the neurotoxins present in the saliva and in the last ten years the sequence of a potential neurotoxin, HT-1, has been determined. In this study we chemically synthesised HT-1 using Boc-chemistry in combination with native chemical ligation. Following successful oxidative folding, we determined the three-dimensional structure of HT-1 by NMR spectroscopy and found a novel structural fold with three of the four disulfide bonds comprising the inhibitory cystine knot (ICK) motif. The fourth disulfide bond connects the second loop to the N-terminal, which decreases the flexibility of the structure. [ABSTRACT FROM AUTHOR]

MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin. [ABSTRACT FROM AUTHOR]

Many insect pests have developed resistance to existing chemical insecticides and consequently there is much interest in the development of new insecticidal compounds with novel modes of action. Although spiders have deployed insecticidal toxins in their venoms for over 250 million years, there is no evolutionary selection pressure on these toxins to possess oral activity since they are injected into prey and predators via a hypodermic needle-like fang. Thus, it has been assumed that spider-venom peptides are not orally active and are therefore unlikely to be useful insecticides. Contrary to this dogma, we show that it is possible to isolate spider-venom peptides with high levels of oral insecticidal activity by directly screening for per os toxicity. Using this approach, we isolated a 34-residue orally active insecticidal peptide (OAIP-1) from venom of the Australian tarantula Selenotypus plumipes. The oral LD50 for OAIP-1 in the agronomically important cotton bollworm Helicoverpa armigera was 104.2±0.6 pmol/g, which is the highest per os activity reported to date for an insecticidal venom peptide. OAIP-1 is equipotent with synthetic pyrethroids and it acts synergistically with neonicotinoid insecticides. The three-dimensional structure of OAIP-1 determined using NMR spectroscopy revealed that the three disulfide bonds form an inhibitor cystine knot motif; this structural motif provides the peptide with a high level of biological stability that probably contributes to its oral activity. OAIP-1 is likely to be synergized by the gut-lytic activity of the Bacillus thuringiensis Cry toxin (Bt) expressed in insect-resistant transgenic crops, and consequently it might be a good candidate for trait stacking with Bt. [ABSTRACT FROM AUTHOR]

Obesity is an increasingly important global health problem that lacks current treatment options. The melanocortin receptor 4 (MC4R) is a target for obesity therapies because its activation triggers appetite suppression and increases energy expenditure. Cyclotides have been suggested as scaffolds for the insertion and stabilization of pharmaceutically active peptides. In this study, we explored the development of appetite-reducing peptides by synthesizing MC4R agonists based on the insertion of the His-Phe-Arg-Trp sequence into the cyclotide kalata B1. The ability of the analogues to fold similarly to kalata B1 but display MC4R activity were investigated. Four peptides were synthesized using t-butoxycarbonyl peptide chemistry with a C-terminal thioester to facilitate backbone cyclization. The structures of the peptides were found to be similar to kalata B1, evaluated by Hα NMR chemical shifts. KB1(GHFRWG;23-28) had a Ki of 29 nM at the MC4R and was 107 or 314 times more selective over this receptor than MC1R or MC5R, respectively, and had no detectable binding to MC3R. The peptide had higher affinity for the MC4R than the endogenous agonist, α-melanocyte stimulation hormone, but it was less potent at the MC4R, with an EC50 of 580 nM for activation of the MC4R. In conclusion, we synthesized melanocortin analogues of kalata B1 that preserve the structural scaffold and display receptor binding and functional activity. KB1(GHFRWG;23-28) is potent and selective for the MC4R. This compound validates the use of cyclotides as scaffolds and has the potential to be a new lead for the treatment of obesity. [ABSTRACT FROM AUTHOR]

The cystine knot is a structural motif that confers exceptional stability on proteins. Here we provide an update on the topology of the cystine knot and the combinatorial diversity of proteins that contain it. We describe recent chemical biology studies that have utilised this structural motif for the development of potential therapeutic or diagnostic agents. The cystine knot appears to have evolved in fungi, plants and animals as a stable and adaptable framework for the display of a wide variety of bioactive peptide sequences, but is amenable to chemical or recombinant synthesis and thus has a wide range of applications in chemistry, biology and medicine. [Copyright &y& Elsevier]

α-Conotoxins are peptides from cone snails that target the nicotinic acetyicholine receptor (nAChR). RgLA and Vcl.1 have analgesic activity in animal pain models. Both peptides target the α9α10 nAChR and inhibit N-type calcium channels via GABAB receptor activation, but the mechanism of action of analgesic activity is unknown. PeIA has previously been shown to inhibit the α9α10 and α3β2 nAChRs. In this study, we have determined the structure of PeIA and shown that it is also a potent inhibitor of N-type calcium channels via GABAB receptor activation. The characteristic α-conotoxin fold is present in PeIA, but it has a different distribution of surface-exposed hydrophobic and charged residues compared with Vcl.1. Thus, the surface residue distribution, rather than the overall fold, appears to be responsible for the 50-fold increase in selectivity at the α3β2 nAChR by PeIA relative to Vcl.1. In contrast to their difference in potency at the nAChR, the equipotent activity of PeIA and Vcl.1 at the GABAB receptor suggests that the GABAB receptor is more tolerant to changes in surface residues than is the nAChR. The conserved Asp-Pro-Arg motif of Vcl.1 and RgIA, which is crucial for potency at the α9α10 nAChR, is not required for activity at GABAB receptor/N-type calcium channels because PeIA has a His-Pro-Ala motif in the equivalent position. This study shows that different structure-activity relationships are associated with the targeting of the GABAB receptor versus nAChRs. Furthermore, there is probably a much more diverse range of conotoxins that target the GABAB receptor than currently realized. [ABSTRACT FROM AUTHOR]

Abstract: Cyclotides are small disulfide-rich peptides that are characterized by a head-to-tail cyclized peptide backbone and a knotted arrangement of three conserved disulfide bonds. They are present in many plants from the Violaceae, Rubiaceae and Cucurbitaceae families, with individual plants expressing a suite of dozens of cyclotides. So far >140 sequences and 15 three-dimensional structures have been determined but it is estimated that the family probably comprises many thousands of members. Their primary function in plants is thought to be as defense agents, based on their potent insecticidal activity, but they also have a range of other biological activities, including anti-HIV, antimicrobial and cytotoxic activities. Because of their exceptional stability they have attracted interest as templates for protein engineering and drug design applications. This article gives an overview of the discovery of cyclotides, describes their unique structural features and range of bioactivities, and discusses their applications in drug design. [Copyright &y& Elsevier]

Conotoxins are small disulfide-rich peptides from the venoms of marine cone snails. They target a variety of ion channels, transporters, and receptors besides the interest in their natural functions in venoms and they are of much interest as drug leads. This short article gives an overview of the structural diversity of conotoxins, and illustrates this diversity with recent selected examples of conotoxin structures. © 2009 IUBMB IUBMB Life, 61(2): 144–150, 2009 [ABSTRACT FROM AUTHOR]

Abstract: The α-conotoxin RgIA is a selective antagonist of the α9α10 nicotinic acetylcholine receptor and has been shown to be a potent analgesic and reduces nerve injury associated inflammation. RgIA was chemically synthesized and found to fold into two disulfide isomers, globular and ribbon. The native globular isomer inhibited ACh-evoked currents reversibly in oocytes expressing rat α9α10 nAChRs but the ribbon isomer was inactive. We determined the three-dimensional structure of RgIA using NMR methods to assist in elucidating the molecular role of RgIA in analgesia and inflammation. [Copyright &y& Elsevier]

Abstract: The cyclotides are macrocyclic knotted proteins characterized by a compact topology and exceptional stability. Accordingly it has been hypothesized that they may be useful as protein engineering frameworks for the stabilization and delivery of bioactive peptide sequences. This study examined the internalization of cyclotides into mammalian cells, a vital step for the delivery of bioactive peptide sequences to intracellular targets. Although the entry of various linear peptides into cells has been reported previously, this is the first report of internalization of a macrocyclic peptide. Cell uptake was examined for representatives of two cyclotide subfamilies; the first was MCoTI-II, a member of the trypsin inhibitor subfamily, which was internalized by a macrophage and breast cancer cell line and the second, the prototypic cyclotide kalata B1 from the Möbius subfamily, which remained extracellular. Biotin labeled MCoTI-II entered macrophages by macropinocytosis, resulting in vesicular encapsulation without trafficking to lysosomes for degradation. The ready uptake, coupled with low cytotoxicity, indicates that MCoTI-II has the potential to transport grafted bioactivities to intracellular targets, making it a potentially valuable framework in drug design applications. [Copyright &y& Elsevier]

Retrocyclins are circular mini-defensins with significant potential as agents against human immunodeficiency virus, influenza A, and herpes simplex virus. Retrocyclins bind carbohydrate-containing surface molecules such as gp120 and CD4 with high affinity (Kd, 10–100 nM), promoting their localization on cell membranes. The structural features important for activity have yet to be fully elucidated, but here, we have determined the first three-dimensional structure of a retrocyclin, namely, one of the most potent forms, retrocyclin-2. In the presence of SDS micelles, a well-defined β-hairpin braced by three disulfide bonds that defines the cystine ladder motif is present. By contrast, a well-defined structure could not be determined in aqueous solution, suggesting that the presence of SDS micelles stabilizes the extended conformation of retrocyclin-2. Translational diffusion measurements indicate that retrocyclin-2 interacts with the SDS micelles, and such a membrane-like interaction may be an important feature in the mechanism of action of these antimicrobial peptides. Analytical ultracentrifugation and the NMR data indicated that retrocyclin-2 self-associates to form a trimer in a concentration-dependent manner. The ability to self-associate may contribute to the high-affinity binding of retrocyclins for glycoproteins by increasing the valency and enhancing the ability of retrocyclins to cross-link cell surface glycoproteins. [ABSTRACT FROM AUTHOR]

α-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell α-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide. [ABSTRACT FROM AUTHOR]

SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI- 1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp14 was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro13 and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active con- formation of the binding loop. [ABSTRACT FROM AUTHOR]

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and non-reducible diselenide bonds. Three isoforms of α-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one ([Sec2,8]ImI or [Sec3,12]ImI), or both ([Sec2,3,8,12]ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native Im!, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the ImI nicotinic acetylcholine receptor, with each seleno-analogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs. [ABSTRACT FROM AUTHOR]

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI–II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys¹–Cys18 disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability. [ABSTRACT FROM AUTHOR]

The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N- and C-termini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity. [Copyright &y& Elsevier]

The plant cyclotides, the largest known family of circular proteins, have tightly folded structures and a range of biological activities that lend themselves to potential pharmaceutical and agricultural applications. Based on sequence homology, they are classified into the bracelet and Möbius subfamilies. The bracelet subfamily has previously been shown to display anti-HIV activity. We show here that a member of the Möbius subfamily, kalata B1, also exhibits anti-HIV activity despite extensive sequence differences between the subfamilies. In addition, acyclic permutants of kalata B1 displayed no anti-HIV activity, suggesting that this activity is critically dependent on an intact circular backbone. [Copyright &y& Elsevier]

α-Conotoxins that target the neuronal nicotinic acetylcholine receptor have a range of potential therapeutic applications and are valuable probes for examining receptor subtype selectivity. The three-dimensional structures of about half of the known neuronal specific α-conotoxins have now been determined and have a consensus fold containing a helical region braced by two conserved disulfide bonds. These disulfide bonds define the two-loop framework characteristic for α-conotoxins, CCXmCXnC, where loop 1 comprises four residues (m = 4) and loop 2 between three and seven residues (n = 3, 6 or 7). Structural studies, particularly using NMR spectroscopy have provided an insight into the role and spatial location of residues implicated in receptor binding and biological activity. [ABSTRACT FROM AUTHOR]

The μO-conotoxins are an intriguing class of conotoxins targeting various voltage-dependent sodium channels and molluscan calcium channels. In the current study, we have shown MrVIA and MrVIB to be the first known peptidic inhibitors of the transient tetrodotoxinresistant (TTX-R) Na+ current in rat dorsal root ganglion neurons, in addition to inhibiting tetrodotoxinsensitive Na+ currents. Human TTX-R sodium channels are a therapeutic target for indications such as pain, highlighting the importance of the μO-conotoxins as potential leads for drug development. Furthermore, we have used NMR spectroscopy to provide the first structural information on this class of conotoxins. MrVIA and MrVIB are hydrophobic peptides that aggregate in aqueous solution but were solubilized in 50% acetonitrile/water. The three-dimensional structure of MrVIB consists of a small β-sheet and a cystine knot arrangement of the three-disulfide bonds. It contains four backbone "loops" between successive cysteine residues that are exposed to the solvent to varying degrees. The largest of these, loop 2, is the most disordered part of the molecule, most likely due to flexibility in solution. This disorder is the most striking difference between the structures of MrVIB and the known δ- and ω-conotoxins, which along with the μO-conotoxins are members of the O superfamily. Loop 2 of ω-conotoxins has previously been shown to contain residues critical for binding to voltage-gated calcium channels, and it is interesting to speculate that the flexibility observed in MrVIB may accommodate binding to both sodium and molluscan calcium channels. [ABSTRACT FROM AUTHOR]

The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities. [Copyright &y& Elsevier]

Biological utilisation of copper requires that the metal, in its ionic forms, be meticulously transported, inserted into enzymes and regulatory proteins, and excess be excreted. To understand the trafficking process, it is crucial that the structures of the proteins involved in the varied processes be resolved. To investigate copper binding to a family of structurally related copper-binding proteins, we have characterised the second Menkes N-terminal domain (MNKr2). The structure, determined using 1H and 15N heteronuclear NMR, of the reduced form of MNKr2 has revealed two α-helices lying over a single β-sheet and shows that the binding site, a Cys(X)2Cys pair, is located on an exposed loop. 1H–15N HSQC experiments demonstrate that binding of Cu(I) causes changes that are localised to conserved residues adjacent to the metal binding site. Residues in this area are important to the delivery of copper by the structurally related Cu(I) chaperones. Complementary site-directed mutagenesis of the adjacent residues has been used to probe the structural roles of conserved residues. [Copyright &y& Elsevier]

Describes the sequences and structures of naturally occurring circular proteins from bacteria. Roles of backbone cyclization; Nuclear magnetic resonance analysis; RNA polymerase; Activities of cyclic bacterial proteins.

Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(2428)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization. [ABSTRACT FROM AUTHOR]

Studies the twists, knots and rings in proteins. Structural definition of the cyclotide framework; Description of the cyclotide family; Characterization of the cyclotide family by a circular protein backbone and conserved cysteine residues linked by disulfide bonds; Provision of the first comprehensive analysis of the topological features in the family of proteins.

Investigates the disulfide folding pathways of cystine knot proteins. Description of the plant cyclotides, a family of circular proteins containing a cyclic knot motif; Isolation and identification of the major folding intermediate; Structural characteristics of the major oxidative folding intermediate of Kalata B.

Presents an overview of the various classes of plant toxins particularly on a class of protein-based plant toxins containing a cystine knot motif. Benefits of protein-based plant toxins for pharmaceutical and agricultural fields; Classes of non-protein plant toxins; Examples of protein-based plant toxins.

Describes the solution structure of BSTI, a trypsin inhibitor from skin secretions of the European frog Bombina bombina. Determination of the disulfide bonds contained by acid proteins; Description of the overall fold of BSTI.

Reports that a series of peptides corresponding to isolated regions of Tau protein have been synthesized and their conformations determined by 1H nuclear magnetic resonance spectroscopy. Immunodominant peptides and a phosphorylated derivative; Recognition by an Alzheimer's disease-specific monoclonal antibody.

Describes the chemical synthesis and folding of the macrocyclic peptide kalata B1. Oxidation of cysteine residues of a linear precursor protein; Cyclization of peptide prior to oxidation; Differences in folding pathways.

Describes the three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor. Sequences of the recombinant repeats; Materials and methods; Comparison of CD spectra; Thrombolytic cleavage; Amide region of one-dimensional 1H spectra of rLB1; Angle order parameters.

We are entering an exciting time in structural biology where artificial intelligence can be used to predict protein structures with greater accuracy than ever before. Extending this level of accuracy to the predictions of disulfide-rich peptide structures is likely to be more challenging, at least in the short term, given the tight packing of cysteine residues and the numerous ways that the disulfide bonds can potentially be linked. It has been previously shown in many cases that several disulfide bond connectivities can be accommodated by a single set of NMR-derived structural data without significant violations. Disulfide-rich peptides are prevalent throughout nature, and arguably the most well-known are those present in venoms from organisms such as cone snails. Here, we have determined the first three-dimensional structure and disulfide connectivity of a U-superfamily cone snail venom peptide, TxVIIB. TxVIIB has a VI/VII cysteine framework that is generally associated with an inhibitor cystine knot (ICK) fold; however, AlphaFold predicted that the peptide adopts a mini-granulin fold with a granulin disulfide connectivity. Our experimental studies using NMR spectroscopy and orthogonal protection of cysteine residues indicate that TxVIIB indeed adopts a mini-granulin fold but with the ICK disulfide connectivity. Our findings provide structural insight into the underlying features that govern formation of the mini-granulin fold rather than the ICK fold and will provide fundamental information for prediction algorithms, as the subtle complexity of disulfide isomers may be not adequately addressed by the current prediction algorithms. [ABSTRACT FROM AUTHOR]

Structural analysis of peptides with nuclear magnetic resonance (NMR) spectroscopy generally relies on knowledge of the primary sequence to enable assignment of the resonances prior to determination of the three-dimensional structure. Resonance assignment without knowledge of the sequence is complicated by redundancy in amino acid type, making complete de novo sequencing using NMR spectroscopy unlikely to be feasible. Despite this redundancy, we show here that NMR spectroscopy can be used to identify short sequence tags that can be used to elucidate full-length peptide sequences via database searching. In the current study, we have used this approach to identify conotoxins from the venom of the cone snail Conus geographus and determined the three-dimensional structure of a member of the I3 superfamily. This approach is most likely to be useful for the characterization of disulfide-rich peptides, such as those that were chosen for this study, as they generally have well-defined structures, which enhances the quality of the NMR spectra. In contrast to other sequencing methods, the lack of sample manipulation, such as protease digestion, allows for subsequent bioassays to be carried out using the native sample used for sequence identification. [ABSTRACT FROM AUTHOR]

Venom‐derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage‐gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNaV1.7 and hCaV3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay‐guided HPLC fractionation revealed a 36‐amino acid peptide with three disulfide bridges named μ/ω‐theraphotoxin‐Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNaV1.7 and hCaV3. Nuclear magnetic resonance structure determination of Pmu1a shows an inhibitor cystine knot fold that is the characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCaV3.2 and hNaV1.7 voltage‐gated channels. [ABSTRACT FROM AUTHOR]

Review: 74 refs. [ABSTRACT FROM AUTHOR]

ApInAPDB (Apoptosis-Inducing Anticancer Peptides Database) consists of 818 apoptosis-inducing anticancer peptides which are manually collected from research articles. The database provides scholars with peptide related information such as function, binding target and affinity, IC50 and etc. In addition, GRAVY (grand average of hydropathy), net charge at pH 7, hydrophobicity and other physicochemical properties are calculated and presented. Another category of information are structural information includes 3D modeling, secondary structure prediction and descriptors for QSAR (quantitative structure–activity relationship) modeling. In order to facilitate the browsing process, three types of user-friendly searching tools are provided: top categories browser, simple search and advanced search. Overall ApInAPDB as the first database presenting apoptosis-inducing anticancer peptides can be useful in the field of peptide design and especially cancer therapy. Researchers can freely access the database at http://bioinf.modares.ac.ir/software/ApInAPDB/. [ABSTRACT FROM AUTHOR]

Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin- 1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP- 1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics. [ABSTRACT FROM AUTHOR]

Peptid mit Schleifchen: Peptidschleifen ‐ Mimetika des klinisch relevanten Norepinephrin ‐ Wiederaufnahmehemmers wurden mithilfe einer Hochdurchsatz ‐ Synthese entwickelt, bei der Peptidthioester zunächst erzeugt und anschließend unter nativer chemischer Ligation cyclisiert werden. Die abgebildeten cyclischen Disulfid ‐ Peptidmimetika sind in Struktur und Funktion dem Stammpeptid sehr ähnlich, aber deutlich stoffwechselbeständiger. [ABSTRACT FROM AUTHOR]

Loopy peptides: Peptide turn mimetics of a clinically relevant norepinephrine reuptake inhibitor were developed employing a high‐throughput synthesis approach to generate peptide thioesters, with subsequent cyclization through native chemical ligation. The vicinal disulfide constrained cyclic peptidomimetics (see scheme) show high structural and functional similarity to the parent peptide, though with superior metabolic stability. [ABSTRACT FROM AUTHOR]

θ-Defensins are ribosomally synthesized cyclic peptides found in the leukocytes of some primate species and have promising applications as antimicrobial agents and scaffolds for peptide drugs. The cyclic cystine ladder motif, comprising a cyclic peptide backbone and three parallel disulfide bonds, is characteristic of θ-defensins. In this study, we explore the role of the cyclic peptide backbone and cystine ladder in the structure, stability, and activity of θ-defensins. θ-Defensin analogues with different numbers and combinations of disulfide bonds were synthesized and characterized in terms of their NMR solution structures, serum and thermal stabilities, and their antibacterial and membrane-binding activities. Whereas the structures and stabilities of the peptides were primarily dependent on the number and position of the disulfide bonds, their antibacterial and membrane-binding properties were dependent on the cyclic backbone. The results provide insights into the mechanism of action of θ-defensins and illustrate the potential of θ-defensin analogues as scaffolds for peptide drug design. [ABSTRACT FROM AUTHOR]

Abstract: The membrane interaction of the cyclotide kalata B1, an all-d-analogue and a single alanine substituted analogue (G6A), was studied by surface plasmon resonance (SPR) and atomic force microscopy (AFM). Kalata B1 showed a strong binding selectivity for dimyristoyl-phosphatidylethanolamine (DMPE) compared to dimyristoyl-phoshatidylcholine (DMPC)-containing lipids. However, when the interaction was visualized by AFM the peptide interacted with DMPC and DMPE in a similar manner. There was no apparent change in membrane morphology with either lipid, suggesting that kalata B1 does not act via a carpet-like disruption mechanism. The d-analogue showed similar binding by SPR and the same strong selectivity for DMPE, indicating that the membrane-interaction and lipid selectivity are not stereo-specific. SPR studies of the G6A analogue revealed that it interacted in a similar way to kalata B1 on the DMPC containing lipids, but showed no increased response on the DMPE containing lipids observed for kalata B1 and d-kalata B1. These results indicate that the Gly6 residue directly influences membrane binding as it is located near a putative membrane interacting hydrophobic patch. Overall, the data suggest that very small changes in amino acid composition (with no change in conformation) can influence specific self-association in combination with membrane binding and mediate the activity of kalata B1. [Copyright &y& Elsevier]

α-Conotoxin RgIA is of interest as a lead in the development of drugs for neuropathic pain. It modulates the α9α10 nicotinic acetylcholine receptor (nAChR) and the GABABreceptor, both of which are implicated in antinociception. However, because of its peptidic nature, RgIA is potentially susceptible to generic problems encountered by peptide-based drugs of poor oral bioavailability, short biological half-life, and low stability. Here, we improved the biopharmaceutical properties of RgIA by backbone cyclization using 3–7 residue peptidic linkers. Cyclization with a six-residue linker does not perturb the overall structure of RgIA, improves selectivity for the GABABreceptor over the α9α10 nAChR, and improves stability in human serum. The results provide insights to further improve the therapeutic properties of RgIA and other conotoxins being considered as drug leads and confirm that cyclization is a readily applicable strategy to improve the stability of peptides with proximate N- and C-termini. [ABSTRACT FROM AUTHOR]

The two disulfide bonds of α-conotoxin ImI, a peptide antagonist of the α7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a γ-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to α-conotoxin ImI by 1H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC50 = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC50 = 6.41 ± 0.09), whereas those of 2 (pIC50 = 5.96 ± 0.09) and 4 (pIC50 = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S═O) to a 3-fold increase (3S═OB) in potencies. [ABSTRACT FROM AUTHOR]

Cyclotides are disulfide-rich peptides from plants that are exceptionally stable as a result of their unique cyclic cystine knot structural motif. Their natural role is thought to be as plant defence agents, most notably against insect pests, but they also have potential applications in drug design and agriculture. This article identifies gaps in current knowledge on cyclotides and suggests future directions for research into this fascinating family of ultra-stable mini-proteins. [ABSTRACT FROM AUTHOR]

Abstract: The beta-arrestins (ARRB1 and ARRB2) regulate G-protein coupled receptor (GPCR) dependent- and independent-signaling pathways and are ubiquitously expressed. Here we show that ARRB2 mRNA and protein expression is enriched in macrophages, and that it regulates complement C1q expression and cell survival. Basal and Toll-like receptor (TLR) inducible expression of mRNAs encoding the complement subcomponents C1qa, C1qb and C1qc was greatly reduced in bone marrow-derived macrophages (BMM) from ARRB2-deficient, but not ARRB1-deficient mice, while factor-independent survival of ARRB2−/− BMM was enhanced compared to wildtype BMM. TatARRB2(23), a cell-permeable peptide that contains the MAPK JNK-binding motif from within the ARRB2 C-domain, impaired ARRB2 interaction with JNK3, down-regulated C1q expression and permitted factor-independent survival in BMM, thus suggesting that this peptide antagonises ARRB2 function in macrophages. In addition, TatARRB2(23) transiently activated the phosphorylation of JNK and ERK, but not p38 in BMM. These data imply that ARRB2 acts to limit JNK/ERK activation and survival in macrophages, but is required for basal and TLR-inducible complement C1q expression. Given that loss of C1q function is strongly associated with the development of systemic lupus erythematosus, ARRB2 may act to limit the development of autoimmune disease. [Copyright &y& Elsevier]

The cyclotides are a large family of circular mini-proteins containing a cystine knot motif. They are expressed in plants as defense-related proteins, with insecticidal activity. Here we investigate their role in membrane interaction and disruption. Kalata B!, a prototypic cyclotide, was found to induce leakage of the self-quenching fluorophore, carboxyfluorescein, from phospholipid vesicles. Alanine-scanning mutagenesis of kalata B! showed that residues essential for lytic activity are clustered, forming a bioactive face. Kalata Bi was sequestered at the membrane surface and showed slow dissociation from vesicles. Electrophysiological experiments showed that conductive pores were induced in liposome patches on incubation with kalata B1. The conductance calculated from the current-voltage relationship indicated that the diameter of the pores formed in the bilayer patches is 4147 Å. Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides. [ABSTRACT FROM AUTHOR]

Relaxin-3 interacts with high potency with three relaxin family peptide receptors (RXFP1, RXFP3, and RXFP4). Therefore, the development of selective agonist and antagonist analogs is important for in vivo studies characterizing the biological significance of the different receptor–ligand systems and for future pharmaceutical applications. Recent reports demonstrated that a peptide selective for RXFP3 and RXFP4 over RXFP1 can be generated by the combination of the relaxin-3 B chain with the A chain from insulin-like peptide 5 (INSL5), creating an R3/I5 chimera. We have used NMR spectroscopy to determine the three-dimensional structure of this peptide to gain structural insights into the consequences of combining chains from two different relaxins. The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B chain compared to native relaxin-3, and the INSL5 A chain displays a relaxin/insulin-like fold with two parallel helices. The findings indicate that binding and activation of RXFP3 and RXFP4 mainly require the B chain and that the A chain functions as structural support. RXFP1, however, demonstrates a more complex binding mechanism, involving both the A chain and the B chain. The creation of chimeras is a promising strategy for generating new structure–activity data on relaxins. [ABSTRACT FROM AUTHOR]