1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 15-23), and two extended strands (residues 24-31 and 34-40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.     

Molecular cloning of cDNA for sapecin and unique expression of the sapecin gene during the development of Sarcophaga peregrina

A cDNA clone for sapecin, an antibacterial protein produced by an embryonic cell line of Sarcophaga peregrina, was isolated and characterized. This clone was found to encode a precursor of sapecin consisting of 94 residues, with sapecin (40 residues) constituting its carboxyl-terminal half. RNA blot hybridization revealed that the gene for the sapecin precursor is activated in the hemocytes of the third instar larvae of Sarcophaga in response to body injury. Thus, sapecin is probably a defense protein synthesized by Sarcophaga to prevent bacterial infection through the damaged body wall. This gene was also found to be activated in the embryonic and early pupal stages, suggesting that sapecin also plays a role in the ontogenetic processes of Sarcophaga.     

Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors

The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.     

Determination of the positions of the disulfide bonds in aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1

Aqualysin I is a heat-stable alkaline serine protease produced by Thermus aquaticus YT-1. Aqualysin I comprises 281 amino acid residues and contains four cysteine residues. The cysteine residues seemed to form disulfide bonds in the molecule. Thus, the positions of the disulfide bonds were investigated. Disulfide bond-containing peptides were identified by peptide mapping with HPLC before and after carboxymethylation of chymotryptic peptides of aqualysin I. The disulfide bond-containing peptides were isolated and then carboxymethylated. Carboxymethylcysteine-containing peptides were purified, and their amino acid compositions and sequences were determined. Based on the data obtained and the primary structure of aqualysin I, it was concluded that two disulfide bonds were formed between Cys67 and Cys99, and between Cys163 and Cys194.     

Channel-forming membrane permeabilization by an antibacterial protein, sapecin: determination of membrane-buried and oligomerization surfaces by NMR

The action mechanism of sapecin, an antibacterial peptide with membrane permeabilization activity, was investigated. The dose dependence of the membrane permeabilization caused by sapecin was sigmoidal, suggesting that sapecin oligomerization leads to the membrane permeabilization. Solution nuclear magnetic resonance analysis of the sapecin-phospholipid vesicle complex revealed the surface buried in the membrane and oligomerization surface on the sapecin molecule. The membrane-buried surface of sapecin was determined by observing the transferred cross-saturation phenomena from the alkyl chains of the phospholipid vesicle to the amide protons of sapecin. The membrane-buried surface contains basic and highly exposed hydrophobic residues, which are suitable for interacting with the acidic bacterial membrane. The oligomerization surface was also identified by comparisons between the results from hydrogen-deuterium exchange experiments and transferred cross-saturation experiments. On the basis of the results from the NMR experiments we built a putative model of sapecin oligomers, which provides insights into the membrane permeabilization caused by insect defensins.     

Disulfide bond formation during the folding of influenza virus hemagglutinin

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.     

Cooperative disulfide bond formation in apamin.

Apamin is being studied as a model for the folding mechanism of proteins whose structures are stabilized by disulfide bonds. Apamin consists of 18 amino acid residues and forms a stable structure consisting of a C-terminal alpha-helix and two reverse turns. This structure is stabilized by two disulfide bonds connecting Cys-1 to Cys-11 and Cys-3 to Cys-15. We used glutathione and dithiothreitol as reference thiols to measure the stabilities of the two disulfide bonds as a function of urea concentration and temperature in order to understand what contributes to the stability of the native structure. The results demonstrate modest contributions from secondary structure to the overall stability of the two disulfide bonds. The equilibrium constants for disulfide bond formation between the fully reduced peptide and the native structure with two disulfide bonds at 25 degrees C and pH 7.0 are 0.42 M2 using glutathione and 2.7 x 10(-5) using dithiothreitol. The equilibrium constant decreases by a factor of approximately 4 in 8 M urea and decreases by a factor of 3 between 0 and 60 degrees C. At least three one-disulfide intermediates are found at low concentrations in the equilibrium mixture. Using glutathione, the equilibrium constants for forming the one-disulfide intermediates with respect to the reduced peptide are approximately 0.025 M. The second disulfide bond forms with an equilibrium constant of approximately 17 M. Thus, apamin folding is very cooperative, but the native structure is only modestly stabilized by urea- or temperature-denaturable secondary structure.     

三叶肽：从结构到功能

三叶结构域是一段由38－39个氨基酸组成的多肽序列,其中包含6个高度保守的半胱氨酸残基,这6个半胱氨酸残基以1－5,2－4,3－6的交联方式形成三对二硫,窝囊鑫肽链折叠成特征性的三叶结构。已发现的哺乳动物三叶肽有三种：pS1、SP及ITF。三叶肽通常位于消化道腔面的粘膜层,具有保护和修复功能,在维持粘膜的完整性中发挥着重要作用。     

Proteinase inhibition by proform of eosinophil major basic protein (pro-MBP) is a multistep process of intra- and intermolecular disulfide rearrangements

The metzincin metalloproteinase pregnancy-associated plasma protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil major basic protein (pro-MBP), which forms a covalent 2:2 proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.     

Location of the disulfide bonds in human plasma prekallikrein: the presence of four novel apple domains in the amino-terminal portion of the molecule

The location of 16 of the 18 disulfide bonds in human plasma prekallikrein was determined by amino acid sequence analysis of cystinyl peptides produced by chemical and enzymatic digestions. A unique structure, named the apple domain, was established for each of the four tandem repeats in the amino-terminal portion of the molecule. The apple domains (90 or 91 amino acids) contain 3 highly conserved disulfide bonds linking the first and sixth, second and fifth, and third and fourth half-cystine residues present in each repeat. The fourth tandem repeat contains an extra disulfide bond that forms a second small loop within the apple domain. The carboxyl-terminal portion of plasma prekallikrein containing the catalytic region of the molecule was found to have disulfide bonds located in positions similar to those of other serine proteases.     

Location of the Disulfide Bonds of the Sweetness-suppressing Polypeptide Gurmarin

The sweetness-suppressing polypeptide gurmarin has been isolated from the leaves of Gymnema sylvestre and consists of 35 amino acid residues including three intramolecular disulfide bonds. The primary structure has already been determined. The positions of the disulfide bonds were located, by a combination of mass spectrometric analysis and sequencing of cystine-containing pep tides obtained by thermolysin-catalyzed hydrolysis of gurmarin, to be at Cys3–Cys18, Cys10–Cys23, and Cys17–Cys33.     

The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism.

The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans. It is a 48-amino acid peptide containing two disulfide bridges, between residues 10 and 48 and 21 and 36, which are crucial for its biological activity. Here, we report the solution structure of STb determined by two- and three-dimensional NMR methods. Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures. The NMR-derived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44. The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb. The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix. The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity. CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function. Reduced STb adopts a predominantly random-coil conformation. Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative. Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds.     

A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin

A new potent antibacterial protein, for which we propose the name royalisin, was found in royal jelly of the honeybee Apis mellifera L. and purified to homogeneity for the first time by acid extraction, gel filtration, and reverse-phase high pressure liquid chromatography. The primary structure of royalisin was determined to consist of 51 residues, with three intramolecular disulfide linkages, having a calculated molecular mass of 5523 Da. Royalisin is an amphipathic protein, with the C-terminal half of the molecule being rich in charged amino acids; and it showed extensive sequence homology to two other antibacterial proteins, sapecin from embryonic Sarcophaga peregrina cells and phormicins from Phormia terranovae larvae. Royalisin was found to have potent antibacterial activity against Gram-positive bacteria at low concentrations, but not against Gram-negative bacteria. Royalisin may be involved in a defense system active against bacterial invasion of the honeybee.     

Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin

Insulin contains three disulfide bonds, one intrachain bond, A6-A11, and two interchain bonds, A7-B7 and A20-B19. Site-directed mutagenesis results (the two cysteine residues of disulfide A7-B7 were replaced by serine) showed that disulfide A7-B7 is crucial to both the structure and activity of insulin. However, chemical modification results showed that the insulin analogs still retained relatively high biological activity when A7Cys and B7Cys were modified by chemical groups with a negative charge. Did the negative charge of the modification groups restore the loss of activity and/or the disturbance of structure of these insulin analogs caused by deletion of disulfide A7-B7? To answer this question, an insulin analog with both A7Cys and B7Cys replaced by Glu, which has a long side-chain and a negative charge, was prepared by protein engineering, and its structure and activity were analyzed. Both the structure and activity of the present analog are very similar to that of the mutant with disulfide A7-B7 replaced by Ser, but significantly different from that of wild-type insulin. The present results suggest that removal of disulfide A7-B7 will result in serious loss of biological activity and the native conformation of insulin, even if the disulfide is replaced by residues with a negative charge.     

Different dynamics and pathway of disulfide bonds reduction of two human defensins,a molecular dynamics simulation study

Human defensins are a class of antimicrobial peptides that are crucial components of the innate immune system. Both human α defensin type 5 (HD5) and human β defensin type 3 (hBD‐3) have 6 cysteine residues which form 3 pairs of disulfide bonds in oxidizing condition. Disulfide bond linking is important to the protein structure stabilization, and the disulfide bond linking and breaking order have been shown to influence protein function. In this project, microsecond long molecular dynamics simulations were performed to study the structure and dynamics of HD5 and hBD‐3 wildtype and analogs which have all 3 disulfide bonds released in reducing condition. The structure of hBD‐3 was found to be more dynamic and flexible than HD5, based on RMSD, RMSF, and radius of gyration calculations. The disulfide bridge breaking order of HD5 and hBD‐3 in reducing condition was predicted by two kinds of methods, which gave consistent results. It was found that the disulfide bonds breaking pathways for HD5 and hBD‐3 are very different. The breaking of disulfide bonds can influence the dimer interface by making the dimer structure less stable for both kinds of defensin. In order to understand the difference in dynamics and disulfide bond breaking pathway, hydrophilic and hydrophobic accessible surface areas (ASA), buried surface area between cysteine pairs, entropy of cysteine pairs, and internal energy were calculated. Comparing to the wildtype, hBD‐3 analog is more hydrophobic, while HD5 is more hydrophilic. For hBD‐3, the disulfide breaking is mainly entropy driven, while other factors such as the solvation effects may take the major role in controlling HD5 disulfide breaking pathway. Proteins 2017; 85:665–681. © 2016 Wiley Periodicals, Inc.     

Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin.

Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.     

G3 domains of aggrecan and PG-M/versican form intermolecular disulfide bonds that stabilize cell-matrix interaction

The extracellular matrix plays a critical role in maintaining tissue integrity. Among the matrix molecules, the large aggregating chondroitin sulfate proteoglycans are the major structural molecules and are the primary contributors to the stability for some tissues such as cartilage. The notable exceptions are nanomelic cartilage and arthritic cartilage: the former contains a point mutation leading to a stop codon before translating to the C-terminal G3 domain; the latter contains a large proportion of aggrecan from which the G3 domain has been cleaved. These phenomena suggest that the G3 domain may be important in cartilage stability. Here, we demonstrated for the first time that the G3 domains of aggrecan and another proteoglycan, PG-M/versican, formed intermolecular disulfide bonds, and all subdomains were involved. Further studies indicated that each of the 10 cysteine residues of the aggrecan G3 domain could potentially form intermolecular disulfide bonds in vitro. The disulfide bonds were disrupted in the presence of reducing reagent beta-mercaptoethanol and dithiothreitol. As a result, normal chondrocyte-matrix interaction was disrupted, and the structure of the extracellular matrix was altered. Furthermore, disruption of disulfide bonds also reduced the role of PG-M/versican G3 domain in mediating cell adhesion. Our study provides strong evidence of the importance of proteoglycan interactions through intermolecular disulfide bonds in cartilage firmness and cell-matrix stability.     

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A.

Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild-type RNase A and each disulfide variant unfold in a two-state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino-acid sequence (Cys26-Cys84 and Cys58-Cys110) enhance stability more than do the two embedded ones (Cys40-Cys95 and Cys65-Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 degrees C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of kcat/Km that are 2- to 40-fold less than that of wild-type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.     

Structural studies of human chorionic gonadotropin and its subunits using tyrosine fluorescence.

The fluorescence properties of the tyrosyl residues of human chorionic gonadotropin (hCG) and its α and β subunits have been examined. The effects of pH, guanidine, and disulfide cleavage on the intensity and polarization of the fluorescence suggest that the isolated subunits possess little, if any, tertiary structure beyond that which is stabilized by the disulfide bonds. Essentially all of the fluorescence of hCG and its subunits was accessible to quenching by iodide ions. Similar results were observed for several other proteins whose fluorescence originates from tyrosyl residues. Thus, we have confirmed and extended the conclusion of R. W. Cowgill ((1966) Biochim. Biophys. Acta120, 196) that the buried tyrosyl residues in ribonuclease fluoresce with a much lower quantum yield than those which are exposed. The dissociation of hCG into its subunits was accompanied by an increase in fluorescence, suggesting the exposure of tyrosyl residues. This was confirmed by difference absorption measurements which indicate a net exposure of two to three tyrosyl residues upon dissociation of the subunits. An additional 0.6 tyrosine was exposed when the disulfide bonds of the β-subunit were cleaved. The polarization of the fluorescence of hCG-β was high (P = 0.19) and, unlike several other proteins with high polarization, could not be lowered by denaturing conditions. Only by cleavage of the disulfide bonds could the fluorescence polarization of either subunit be lowered to a value (P = 0.08) characteristic of a random polypeptide. It appears that the disulfide bonds play an important role in maintaining the rigidity of the fluorescent tyrosyl residues, located at or near the surface of the protein.     

Determination of the three-dimensional structure of iberiotoxin in solution by 1H nuclear magnetic resonance spectroscopy.

The solution structure of chemically synthesized iberiotoxin, a scorpion toxin that blocks Ca(2+)-activated K+ channels, has been determined using 2D 1H NMR spectroscopy. Analysis of the NOEs, coupling constants, and HN-DN exchange rates indicates the structure consists of an antiparallel beta-sheet from residues 25 to 36, with a type 1 turn at residues 30-31, and a helix from residues 13 to 21. The carboxyl-terminal residues form a short, and distorted, third strand of the sheet. The NMR data are consistent with disulfide bonds from residues 7 to 28, 13 to 33, and 17 to 35. The disulfide bridging presents the same profile as in other scorpion toxins, where a Cys-X-Cys sequence in a strand of sheet forms two disulfide bonds to a Cys-X-X-X-Cys sequence in a helix. Three-dimensional structures were generated using the torsion angle space program PEGASUS. The best ten structures had an average rmsd over all pairwise comparisons of 1.49 A. The average rmsd to a calculated average structure is 1.0 A. The resulting structures appear very similar to those of charybdotoxin, a related scorpion toxin.