Disulfide Connectivity Research Articles

Im10A, a short conopeptide isolated from Conus imperialis and possesses two highly concentrated disulfide bridges and analgesic activity

In the present study, we isolated, synthesized and NMR structurally characterized a novel conopeptide Im10A consisting of 11 amino acids (NTICCEGCMCY-NH2) from Conus imperialis. Unlike other conopeptides with four cysteine residues, Im10A had only two residues in loop 1 and one residue in loop 2 (CC-loop1-C-loop2-C), which formed a stable disulfide connectivity “I-IV, II- III” (framework X) with a type I β-turn. Interestingly, Im10A exhibited 50.7% analgesic activity on rat partial sciatic nerve ligation (PNL) at 2h after Im10A administration. However, 10μM Im10A exhibited no apparent effect on neuronal nicotinic acetylcholine receptor, and it did not target DRG voltage-dependent sodium, potassium and calcium ion channels and opioid receptor. To our knowledge, Im10A had the most concentrated disulfide bridges among conopeptides with four cysteine residues. This finding provided a new motif for the future development of biomimetic compounds.

A novel 4/6-type alpha-conotoxin ViIA selectively inhibits nAchR α3β2 subtype.

Conotoxins (CTxs) are typically small peptides composed of 12-50 amino acid residues with 2-5 disulfide bridges. Most of them potently and selectively target a wide variety of ion channels and membrane receptors. They are highly valued as neuropharmacological probes and in pharmaceutical development. In this work, a novel α4/6-CTx named ViIA (RDCCSNPPCAHNNPDC-NH2) was identified from a cDNA library of the venom ducts of Conus virgo (C. virgo). ViIA was then synthesized chemically and its disulfide connectivity was identified as 'C(1)-C(3), C(2)-C(4)'. Its molecular targets were further assessed using two-electrode voltage clamping. The results indicated that ViIA selectively inhibited nicotinic acetylcholine receptor (nAChR) α3β2 subtype with an IC50 of 845.5 nM, but did not target dorsal root ganglion sodium (Na(+))-, potassium (K(+))- or calcium (Ca(2+))-ion channels. Further structure-activity relationship analysis demonstrated that Arg(1) and His(11) but not Asp(2) were the functional residues. To the best of our knowledge, ViIA is the first 4/6 α-CTx that selectively inhibits nAChR α3β2 subtype. This finding expands the knowledge of targets of α4/6-family CTxs.

Accurate disulfide-bonding network predictions improve ab initio structure prediction of cysteine-rich proteins.

Cysteine-rich proteins cover many important families in nature but there are currently no methods specifically designed for modeling the structure of these proteins. The accuracy of disulfide connectivity pattern prediction, particularly for the proteins of higher-order connections, e.g., >3 bonds, is too low to effectively assist structure assembly simulations. We propose a new hierarchical order reduction protocol called Cyscon for disulfide-bonding prediction. The most confident disulfide bonds are first identified and bonding prediction is then focused on the remaining cysteine residues based on SVR training. Compared with purely machine learning-based approaches, Cyscon improved the average accuracy of connectivity pattern prediction by 21.9%. For proteins with more than 5 disulfide bonds, Cyscon improved the accuracy by 585% on the benchmark set of PDBCYS. When applied to 158 non-redundant cysteine-rich proteins, Cyscon predictions helped increase (or decrease) the TM-score (or RMSD) of the ab initio QUARK modeling by 12.1% (or 14.4%). This result demonstrates a new avenue to improve the ab initio structure modeling for cysteine-rich proteins. http://www.csbio.sjtu.edu.cn/bioinf/Cyscon/ zhng@umich.edu or hbshen@sjtu.edu.cn. Supplementary data are available at Bioinformatics online.

An Evolutionary View on Disulfide Bond Connectivities Prediction Using Phylogenetic Trees and a Simple Cysteine Mutation Model.

Disulfide bonds are crucial for many structural and functional aspects of proteins. They have a stabilizing role during folding, can regulate enzymatic activity and can trigger allosteric changes in the protein structure. Moreover, knowledge of the topology of the disulfide connectivity can be relevant in genomic annotation tasks and can provide long range constraints for ab-initio protein structure predictors. In this paper we describe PhyloCys, a novel unsupervised predictor of disulfide bond connectivity from known cysteine oxidation states. For each query protein, PhyloCys retrieves and aligns homologs with HHblits and builds a phylogenetic tree using ClustalW. A simplified model of cysteine co-evolution is then applied to the tree in order to hypothesize the presence of oxidized cysteines in the inner nodes of the tree, which represent ancestral protein sequences. The tree is then traversed from the leaves to the root and the putative disulfide connectivity is inferred by observing repeated patterns of tandem mutations between a sequence and its ancestors. A final correction is applied using the Edmonds-Gabow maximum weight perfect matching algorithm. The evolutionary approach applied in PhyloCys results in disulfide bond predictions equivalent to Sephiroth, another approach that takes whole sequence information into account, and is 26–29% better than state of the art methods based on cysteine covariance patterns in multiple sequence alignments, while requiring one order of magnitude fewer homologous sequences (103 instead of 104), thus extending its range of applicability. The software described in this article and the datasets used are available at http://ibsquare.be/phylocys.

A Disulfide Stabilized β-Sandwich Defines the Structure of a New Cysteine Framework M-Superfamily Conotoxin.

The structure of a new cysteine framework (-C-CC-C-C-C-) "M"-superfamily conotoxin, Mo3964, shows it to have a β-sandwich structure that is stabilized by inter-sheet cross disulfide bonds. Mo3964 decreases outward K(+) currents in rat dorsal root ganglion neurons and increases the reversal potential of the NaV1.2 channels. The structure of Mo3964 (PDB ID: 2MW7 ) is constructed from the disulfide connectivity pattern, i.e., 1-3, 2-5, and 4-6, that is hitherto undescribed for the "M"-superfamily conotoxins. The tertiary structural fold has not been described for any of the known conus peptides. NOE (549), dihedral angle (84), and hydrogen bond (28) restraints, obtained by measurement of (h3)JNC' scalar couplings, were used as input for structure calculation. The ensemble of structures showed a backbone root mean square deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (ϕ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules.

Topological Complexity in Protein Structures

Abstract For DNA molecules, topological complexity occurs exclusively as the result of knotting or linking of the polynucleotide backbone. By contrast, while a few knots and links have been found within the polypeptide backbones of some protein structures, non-planarity can also result from the connectivity between a polypeptide chain and inter- and intra-chain linking via cofactors and disulfide bonds. In this article, we survey the known types of knots, links, and non-planar graphs in protein structures with and without including such bonds and cofactors. Then we present new examples of protein structures containing Möbius ladders and other non-planar graphs as a result of these cofactors. Finally, we propose hypothetical structures illustrating specific disulfide connectivities that would result in the key ring link, the Whitehead link and the 51 knot, the latter two of which have thus far not been identified within protein structures.

Disulfide Connectivity Prediction Based on Modelled Protein 3D Structural Information and Random Forest Regression.

Disulfide connectivity is an important protein structural characteristic. Accurately predicting disulfide connectivity solely from protein sequence helps to improve the intrinsic understanding of protein structure and function, especially in the post-genome era where large volume of sequenced proteins without being functional annotated is quickly accumulated. In this study, a new feature extracted from the predicted protein 3D structural information is proposed and integrated with traditional features to form discriminative features. Based on the extracted features, a random forest regression model is performed to predict protein disulfide connectivity. We compare the proposed method with popular existing predictors by performing both cross-validation and independent validation tests on benchmark datasets. The experimental results demonstrate the superiority of the proposed method over existing predictors. We believe the superiority of the proposed method benefits from both the good discriminative capability of the newly developed features and the powerful modelling capability of the random forest. The web server implementation, called TargetDisulfide, and the benchmark datasets are freely available at: http://csbio.njust.edu.cn/bioinf/TargetDisulfide for academic use.

Allotides: Proline-Rich Cystine Knot α-Amylase Inhibitors from Allamanda cathartica.

Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.

One short cysteine-rich sequence pattern - two different disulfide-bonded structures - a molecular dynamics simulation study.

The nematocyst walls of Hydra are formed by proteins containing small cysteine-rich domains (CRDs) of ~25 amino acids. The first CRD of nematocyst outer all antigen (NW1) and the C-terminal CRD of minicollagen-1 (Mcol1C) contain six cysteines at identical sequence positions, however adopt different disulfide bonded structures. NW1 shows the disulfide connectivities C2-C14/C6-C19/C10-C18 and Mcol1C C2-C18/C6-C14/C10-C19. To analyze if both show structural preferences in the open, non-disulfide bonded form, which explain the formation of either disulfide connectivity pattern, molecular dynamics (MD) simulations at different temperatures were performed. NW1 maintained in the 100-ns MD simulations at 283 K a rather compact fold that is stabilized by specific hydrogen bonds. The Mcol1C structure fluctuated overall more, however stayed most of the time also rather compact. The analysis of the backbone Φ/ψ angles indicated different turn propensities for NW1 and Mcol1C, which mostly can be explained based on published data about the influence of different amino acid side chains on the local backbone conformation. Whereas a folded precursor mechanism may be considered for NW1, Mcol1C may fold according to the quasi-stochastic folding model involving disulfide bond reshuffling and conformational changes, locking the native disulfide conformations. The study further demonstrates the power of MD simulations to detect local structural preferences in rather dynamic systems such as the open, non-disulfide bonded forms of NW1 and Mcol1C, which complement published information from NMR backbone residual dipolar couplings. Because the backbone structural preferences encoded by the amino acid sequence embedding the cysteines influence which disulfide connectivities are formed, the data are generally interesting for a better understanding of oxidative folding and the design of disulfide stabilized therapeutics.

Cystamindi-ium tetrachlorocuprate [NH3(CH2)2SS(CH2)2NH3][CuCl4]: synthesis, crystal structure, and thermal decomposition

The salt [NH3(CH2)2SS(CH2)2NH3][CuCl4] was obtained by crystallization after adding CuCl2 to cystamine (Cysta), solved in hydrochloric acid. The assumption of conserved disulfide connection (S–S) in the compound, made on the basis of infrared spectroscopy, is further supported by the crystal structure determined from X-ray powder diffraction data. The compound has an ionic structure. [CuCl4]2− and CystaH22+ ions package in the form of inorganic and organic layers in the cell, interconnected through the formation of hydrogen bonds via NH3-groups and chlorine atoms of the complex [CuCl4]2−. Inorganic layers are additionally stabilized in the parquet package of [CuCl4]2− ions which provides a Cu-distorted octahedral coordination. CystaH2[CuCl4] is stable in air up to 200 °C. Thermal decomposition occurs in several stages, accompanied by breaking of S–S bonds, releasing of the organic component and yielding CuO.

Soft Computing Methods for Disulfide Connectivity Prediction

The problem of protein structure prediction (PSP) is one of the main challenges in structural bioinformatics. To tackle this problem, PSP can be divided into several subproblems. One of these subproblems is the prediction of disulfide bonds. The disulfide connectivity prediction problem consists in identifying which nonadjacent cysteines would be cross-linked from all possible candidates. Determining the disulfide bond connectivity between the cysteines of a protein is desirable as a previous step of the 3D PSP, as the protein conformational search space is highly reduced. The most representative soft computing approaches for the disulfide bonds connectivity prediction problem of the last decade are summarized in this paper. Certain aspects, such as the different methodologies based on soft computing approaches (artificial neural network or support vector machine) or features of the algorithms, are used for the classification of these methods.

De novo discovery of bioactive cyclic peptides using bacterial display and flow cytometry.

Cyclic peptides are increasingly desired for their enhanced stability and pharmacologic properties. Due to their limited conformational flexibility, cyclic peptides with C-to-N-terminal peptide bond and a disulfide bridge can confer high target binding affinity and resistance to proteolytic enzymes. Challenging drug targets including protein interaction surfaces can be successfully targeted using peptides rather than small molecules or proteins. Peptides, capable of antibody-like affinities with increased potency, can be designed to fill in the gap between small molecules and larger proteins. However, cysteine-rich peptides with several disulfide bonds have limitations in production and purification. Therefore, we devised a strategy to identify cyclic peptides with single disulfide connectivity that offers desired properties along with ease in synthesis and production. Here, de novo design of cyclic peptides is demonstrated through screening of peptide libraries using bacterial display and cell sorting. Herein, a step-by-step protocol is presented to design and screen diverse peptide libraries to identify cyclic peptides with desired specificity and affinity towards arbitrary target proteins.

The identification of disulfides in ricin D using proteolytic cleavage followed by negative-ion nano-electrospray ionization mass spectrometry of the peptide fragments.

To use negative-ion nano-electrospray ionization mass spectrometry of peptides from the tryptic digest of ricin D, to provide sequence information; in particular, to identify disulfide position and connectivity. Negative-ion fragmentations of peptides from the tryptic digest of ricin D was studied using a Waters QTOF2 mass spectrometer operating in MS and MS(2) modes. Twenty-three peptides were obtained following high-performance liquid chromatography and studied by negative-ion mass spectrometry covering 73% of the amino-acid residues of ricin D. Five disulfide-containing peptides were identified, three intermolecular and two intramolecular disulfide-containing peptides. The [M-H](-) anions of the intermolecular disulfides undergo facile cleavage of the disulfide units to produce fragment peptides. In negative-ion collision-induced dissociation (CID) these source-formed anions undergo backbone cleavages, which provide sequencing information. The two intramolecular disulfides were converted proteolytically into intermolecular disulfides, which were identified as outlined above. The positions of the five disulfide groups in ricin D may be determined by characteristic negative-ion cleavage of the disulfide groups, while sequence information may be determined using the standard negative-ion backbone cleavages of the resulting cleaved peptides. Negative-ion mass spectrometry can also be used to provide partial sequencing information for other peptides (i.e. those not containing Cys) using the standard negative-ion backbone cleavages of these peptides.

Clustering-based model of cysteine co-evolution improves disulfide bond connectivity prediction and reduces homologous sequence requirements.

Cysteine residues have particular structural and functional relevance in proteins because of their ability to form covalent disulfide bonds. Bioinformatics tools that can accurately predict cysteine bonding states are already available, whereas it remains challenging to infer the disulfide connectivity pattern of unknown protein sequences. Improving accuracy in this area is highly relevant for the structural and functional annotation of proteins. We predict the intra-chain disulfide bond connectivity patterns starting from known cysteine bonding states with an evolutionary-based unsupervised approach called Sephiroth that relies on high-quality alignments obtained with HHblits and is based on a coarse-grained cluster-based modelization of tandem cysteine mutations within a protein family. We compared our method with state-of-the-art unsupervised predictors and achieve a performance improvement of 25-27% while requiring an order of magnitude less of aligned homologous sequences (∼10(3) instead of ∼10(4)). The software described in this article and the datasets used are available at http://ibsquare.be/sephiroth. wvranken@vub.ac.be Supplementary material is available at Bioinformatics online.

Novel insights into structure and function of factor XIIIa-inhibitor tridegin.

The inhibition of the final step in blood coagulation, the factor XIIIa (FXIIIa) catalyzed cross-linking of fibrin monomers, is currently still a challenge in medicinal chemistry. We report synthesis, recombinant expression, disulfide connectivity, and biological activity of tridegin, the sole existing peptide representative displaying inhibitory activity on FXIIIa. Inhibition of the enzyme by this 66-mer cysteine-rich peptide is mediated by its C-terminal sequence, while the N-terminal part comprises structural information and contributes to inhibitor binding. Either of the production strategies examined leads to the formation of different disulfide-bridged isomers indicating the requirement of the correct fold for inhibitory activity. Molecular modeling and docking studies confirm disulfide bond isomer preference with respect to binding to FXIIIa, in turn, the knowledge of the enzyme-inhibitor interactions might bring about comprehensive ideas for the design of a suitable lead structure for addressing FXIIIa.

Copsin, a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/β-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.

Structure-activity relationships of the intramolecular disulfide bonds in coprisin, a defensin from the dung beetle

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630]

The intramolecular disulfide‐stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin‐like structural module

The growing emergence of antibiotic-resistant bacteria has led to the exploration of naturally occurring defense peptides as antimicrobials. In this study, we found that laterosporulin (LS), a class IId bacteriocin, effectively kills active and nonmultiplying cells of both Gram-positive and Gram-negative bacteria. Fluorescence and electron microscopy suggest that growth inhibition occurs because of increased membrane permeability. The crystal structure of LS at 2.0 Å resolution reveals an all-β conformation of this peptide, with four β-strands forming a twisted β-sheet. All six intrinsic cysteines are intramolecularly disulfide-bonded, with two disulfides constraining the N terminus of the peptide and the third disulfide crosslinking the extreme C terminus, resulting in the formation of a closed structure. The significance of disulfides in maintaining the in-solution peptide structure was confirmed by CD and fluorescence analyses. Despite a low overall sequence similarity, LS has disulfide connectivity [C(I)-C(V), C(II)-C(IV), and C(III)-C(VI)] like that of β-defensins and a striking architectural similarity with α-defensins. Therefore LS presents a missing link between bacteriocins and mammalian defensins, and is also a potential antimicrobial lead, in particular against nonmultiplying bacteria.

Application of room-temperature aprotic and protic ionic liquids for oxidative folding of cysteine-rich peptides.

The oxidation of the conotoxin μ-SIIIA in different ionic liquids was investigated, and the results were compared with those obtained in [C2 mim][OAc]. Conversion of the reduced precursor into the oxidized product was observed in the protic ILs methyl- and ethylammonium formate (MAF and EAf, respectively), whereas choline dihydrogenphosphate and Ammoeng 110 failed to yield folded peptide. However, the quality and yield of the peptide obtained in MAF and EAF were lower than in the case of the product from [C2 mim][OAc]. Reaction conditions (temperature, water content) also had an impact on peptide conversion. A closer look at the activities of μ-SIIIA versions derived from an up-scaled synthesis in [C2 mim][OAc] revealed a significant loss of the effect on ion channel NaV 1.4 relative to the buffer-oxidized peptide, whereas digestion of either μ-SIIIA product by trypsin was unaffected. This was attributed to adherence of ions from the IL to the peptide, because the disulfide connectivity is basically the same for the differentially oxidized μ-SIIIA versions.

Protected hinge in the immunoglobulin G2-A2 disulfide isoform.

Human IgG2 consists of disulfide-mediated structural isoforms, classified by the number of Fab arms disulfide-linked to the heavy chain hinge. In the IgG2-B isoform, both Fab arms are linked to the hinge region, and in IgG2-A, neither Fab arm are linked to the hinge. IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide-linked to the hinge. Within each of these isoform types are subtypes, with subtle disulfide-linkage differences. Here we explored the structural basis for the A1 and A2 isoform subtypes. Whereas A1 isoform converts into the A/B and B isoforms under mild redox conditions, A2 does not. Characterization of the disulfide connectivities of A2 isoform revealed a similar structure to A1 isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A2 isoform were resistant to reduction under conditions where A1 isoform hinge disulfides became reduced and they required thermal treatment (>55 °C) to obtain thiol-dependent disulfide reduction. Structural analysis of the hinge region indicated that the protected disulfides were restricted to cysteines 219 and 220 of the upper hinge. Disruption of the upper hinge through insertion mutagenesis eliminated A2 isoform behavior. (1)H NMR studies showed that the A1 isoform Fc glycan was more dynamic than that on A2 isoform and showed some other conformational differences. Results point to an IgG2-A2 upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG.