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.     

Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli

To determine the modes of three disulfide linkages in the heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli, we synthesized STh(6-18), which consists of 13 amino acid residues and has the same intramolecular disulfide linkages as native STh [(1985) FEBS Lett. 181, 138-142], by stepwise and selective formation of disulfide bonds using different types of removable protecting groups for the Cys residues. Synthesis of the peptide with different modes of disulfide bond formation provided three peptides consistent with standard STh(6-18) in their physicochemical and biological properties, thereby indicating that the disulfide bonds in STh(6-18) are Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys.     

On the structural stability of a small bioactive peptide of potential use in biotechnology.

A tridecapeptide with the sequence CCEICCNPACFGC has been synthesized to reproduce the active moiety of a heat stable enterotoxin from Vibrio cholerae. The proton NMR analysis indicates, for the active synthetic fragment, a rigid secondary structure stabilised by three disulfide bridges. Such a rigid peptide, suitably detoxified and activated, could be a good candidate to be used as a carrier for linear bioactive peptides or other functional groups.     

Improved method for purification of human Escherichia coli heat-stable enterotoxin by hydrophobic interaction, molecular-sieve and high performance ion exchange chromatography

A heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli has been purified to homogeneity by hydrophobic interaction, molecular-sieve and high performance liquid chromatography with a recovery of 48%. The toxin is composed of 10 different amino acids with a total of 18 amino acid residues, one-third of which are half-cystine. Purified enterotoxin contains no carbohydrate and is biologically active in the suckling mouse test in 2.1-ng quantities. The molecule was heat-stable (80 degrees C, 20 min.) at pH 7 and pH 2 but lost biological activity at pH 12. Biological activity was lost when treated with the reducing agent dithiothreitol, suggesting that the presence of disulfide bridges is required for biological activity.     

Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-gamma was studied by reversed-phase HPLC. IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule.     

Improved Method for Purification of Human Escherichia Col I Heat-Stable Enterotoxin by Hydrophobic Interaction,Molecular-Sieve and High Performance Ion Exchange Chromatography

Abstract

A heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli has been purified to homogeneity by hydrophobic interaction, molecular-sieve and high performance liquid chromatography with a recovery of 48%. The toxin is composed of 10 different amino acids with a total of 18 amino acid residues, one-third of which are half-cystine. Purified enterotoxin contains no carbohydrate and is biologically active in the suckling mouse test in 2.1-ng quantities. The molecule was heat-stable (80 C, 20 min.) at pH 7 and pH 2 but lost biological activity at pH 12. Biological activity was lost when treated with the reducing agent dithiothreitol, suggesting that the presence of disulfide bridges is required for biological activity.     

Truncated atrial natriuretic factor analogs retain full agonist activity.

The synthesis, receptor binding, and agonist activity of a series of truncated atrial natriuretic analogs (ANF) are described. These analogs incorporate two portions of the native 28 amino peptide, the eight amino acids C-terminal to Cys7, and two amino acids from the C-terminus (phenylalanine and arginine), into disulfide-bonded cyclic peptides. The inclusion of the C-terminal amino acids converted the ANF analogs from receptor ligands to full agonists, as measured by several methods, including the stimulation of cGMP biosynthesis in endothelial cells, inhibition of aldosterone biosynthesis in rat adrenal cells, and natriuretic-hypotensive activity in vivo. The most potent analogs have cyclohexylalanine (Cha) at position 8. The lead compound (Arg6,Cha8 ANF 6-15 Phe-Arg-Cys-NH2) is a tridecapeptide that integrates the C-terminal amino acids inside the disulfide ring. This peptide, designated as A-68828, has a binding affinity of IC50 = 120 nM, approximately 1/400 of ANF 1-28. However, this analog, in vivo, is only slightly less natriuretic (1/20-1/50) than ANF 1-28. Unlike the native peptide, A-68828 is only mildly hypotensive and at the highest concentration tested reduced blood pressure less than 15 mmHg (1 mmHg = 133.322 Pa). A-68828 inhibited ACTH-induced aldosterone release to a greater extent than ANF 1-28: 100 vs. 50%. The selective natriuretic activity of A-66828, relative to ANF, suggests clinical utility for the treatment of acute renal failure.     

Designed peptide analogues of the potassium channel blocker ShK toxin.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35-residue polypeptide cross-linked by 3 disulfide bridges. In an effort to generate truncated peptidic analogues of this potent channel blocker, we have evaluated three analogues, one in which the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges), and two in which non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. The effect of introducing a lactam bridge in the first helix of ShK toxin (to create cyclo14/18[Lys14,Asp18]ShK) was also examined to confirm that this modification was compatible with activity. All four analogues were tested in vitro for their ability to block Kv1.3 potassium channels in Xenopus oocytes, and their solution structures were determined using 1H NMR spectroscopy. The lactam bridge in full-length ShK is well tolerated, with only a 5-fold reduction in binding to Kv1.3. The truncated and stabilized analogue was inactive, apparently due to a combination of slight deviations from the native structure and alterations to side chains required for binding. One of the peptide scaffolds was also inactive because it failed to adopt the required structure, but the other had a K(d) of 92 microM. This active peptide incorporated mimics of Lys22 and Tyr23, which are essential for activity in ShK, and an Arg residue that could mimic Arg11 or Arg24 in the native toxin. Modification of this peptide should produce a more potent, low molecular weight peptidic analogue which will be useful not only for further in vitro and in vivo studies of the effect of blocking Kv1.3, but also for mapping the interactions with the pore and vestibule of this K(+) channel that are required for potent blockade.     

Evolutionary origin of pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae O1.

Three families of the evolutionarily related pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae O1, a family of cholera enterotoxin (CT) and heat-labile enterotoxin (LT) including CT, LTh, and LTp, a family of heat-stable enterotoxin I (STI) including STIa and STIb, and a family of K88 enteroadhesion fimbriae including K88ab, K88ac, and K88ad were analyzed for synonymous (silent) nucleotide substitutions by using the gene nucleotide sequences of earlier reports and the LTp gene nucleotide sequence presented in this paper. The data suggested that the divergences between LT and CT and between STIa and STIb occurred in the remote past, whereas those between LTh and LTp and between members of the K88 family occurred very recently. We concluded that the LT gene is a foreign gene that has been acquired by E. coli to form an enteropathogen. This provides evolutionary evidence of species-to-species transfer of pathogenic determinants in procaryotes.     

Role of disulfide bridges in the folding, structure and biological activity of omega-conotoxin GVIA.

Omega-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the cone shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by (1)H NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges were synthesised using orthogonal protection of cysteine residues. In the first analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing the appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was detected for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five inactive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379-384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to the disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which presumably accounts for their lack of activity. The NMR studies also showed that all five analogues had substantially altered tertiary structures, although the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogues despite the non-native disulfide pairings. This work demonstrates the importance of the disulfides in omega-conotoxin folding and shows that the Cys-15-Cys-26 disulfide is essential for activity in GVIA. The NMR analyses also emphasise that backbone chemical shifts and short- and medium-range NOEs are dictated largely by local secondary structure elements and are not necessarily reliable monitors of the tertiary fold.     

Mutations in Rotavirus Nonstructural Glycoprotein NSP4 Are Associated with Altered Virus Virulence

Rotaviruses are major pathogens causing life-threatening dehydrating gastroenteritis in children and animals. One of the nonstructural proteins, NSP4 (encoded by gene 10), is a transmembrane, endoplasmic reticulum-specific glycoprotein. Recently, our laboratory has shown that NSP4 causes diarrhea in 6- to 10-day-old mice by functioning as an enterotoxin. To confirm the role of NSP4 in rotavirus pathogenesis, we sequenced gene 10 from two pairs of virulent and attenuated porcine rotaviruses, the OSU and Gottfried strains. Comparisons of the NSP4 sequences from these two pairs of rotaviruses suggested that structural changes between amino acids (aa) 131 and 140 are important in pathogenesis. We next expressed the cloned gene 10 from the OSU virulent (OSU-v) and OSU attenuated (OSU-a) viruses by using the baculovirus expression system and compared the biological activities of the purified proteins. NSP4 from OSU-v virus increased intracellular calcium levels over 10-fold in intestinal cells when added exogenously and 6-fold in insect cells when expressed endogenously, whereas NSP4 from OSU-a virus had little effect. NSP4 from OSU-v caused diarrhea in 13 of 23 neonatal mice, while NSP4 from OSU-a caused disease in only 4 of 25 mice (P < 0.01). These results suggest that avirulence is associated with mutations in NSP4. Results from site-directed mutational analyses showed that mutated OSU-v NSP4 with deletion or substitutions in the region of aa 131 to 140 lost its ability to increase intracellular calcium levels and to induce diarrhea in neonatal mice, confirming the importance of amino acid changes from OSU-v NSP4 to OSU-a NSP4 in the alteration of virus virulence.     

A Molecular Dynamics Study of Human Defensins HBD-1 and HNP-3 in Water

Abstract

Mammalian defensins are crucial components of the innate immune system. They are characterized by three disulfide bridges and exhibit broad spectrum antibacterial activity. The spacing between the cysteines and disulfide connectivities in the two classes of defensins, the α- and β-forms, are different. The structural motif of 3 β-strands appears to be conserved in α and β-defensins despite differences in disulfide connectivities and spacing between cysteines. In this study, Molecular Dynamics Simulations (MDS) have been carried out to study the conformational behavior of α- and β-defensins with and without disulfide bridges. Our results indicate that β-strands in the C-terminal region of HBD-1 and HNP-3 do not unfold during the course of MDS. The segment adopting α-helix in HBD-1 unfolds early during the simulations. The backbone hydrogen bonds in HBD-1 and HNP-3 are broken during MDS. When the disulfide bonds are absent, the N-terminal β-strand unfolds by 20 ns but β-strands are observed in the C-terminal region of HNP-3. HBD-1, without disulfide bridges, unfolds to a greater extent during the course of the MDS. Examination of distances between sulfur atoms of cysteines without disulfide bridges during the simulations indicate that there is no specific preference for native disulfide bridges, which could be the reason for the experimental observation of non-native disulfide bridge formation during chemical synthesis of human α- and β-defensins. Since defensins with non-native disulfide bridges are biologically active, the exact three dimensional structures observed for native HBD-1 and HNP-3 does not appear to be essential for exhibiting antibacterial activity.     

Structure/activity relationships of C-terminal neuropeptide Y peptide segments and analogues composed of sequence 1-4 linked to 25-36

C-terminal analogues of neuropeptide Y have been synthesized. The influence of chain length, single-amino-acid substitutions and segment substitutions on receptor binding, biological activity and conformational properties has been investigated. Receptor binding and in vivo assays revealed biological activity already for amino acids 28-36 of neuropeptide Y [neuropeptide Y-(Ac-28-36)-peptide] which increased with increasing chain length. Replacement of Arg25 in neuropeptide Y-(Ac-25-36)-peptide had no influence on binding, whereas Arg33 and Arg35 cannot be replaced by lysine or ornithine without considerable decrease in receptor binding. The introduction of conformational constraints by the 2-aminoisobutyric acid residue (Aib) in position 30 and replacing the amino acids 28-32 by Ala-Aib-Ala-Aib-Ala decreased receptor binding. However, the corresponding Aib-Ala-Aib-Ala-Aib-substituted analogue and a more flexible analogue with Gly5 at position 28-32 exhibited considerable affinity for the receptor. All these substitutions led to a decrease in postsynaptic activity. Strong agonistic activities could be detected in a series of 10 discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was neuropeptide Y amino acids 1-4 linked to amino acids 25-36 through aminohexanoic acid (Ahx) [neuropeptide Y-(1-4-Ahx-25-36)-peptide].     

Studies on the total synthesis of an A7,B7-dicarbainsulin. III. Assembly of segments and generation of biological activity

As a further contribution to the synthesis of an insulin analogue with a stable A7-B7 interchain bond, the synthesis of A(8-21) by solution methods, and of B(9-25) as well as [7-(2,7-diaminosuberic acid)]B(1-8) by solid phase methods is described. In the latter compound, the amino group of the diaminosuberic acid residue was acylated with A(1-6), and the resulting "U-peptide" sequentially elongated with the C-terminal A- and finally B-chain sequences. The conversion of the product into the disulfide moiety gave a mixture which could not be resolved by currently available methods. However, the low biological activity of the crude product indicates that the A7-B7 disulfide bond is not crucially important for the activity of insulin.     

Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of Escherichia coli. A putative binding site for a binding protein on rat intestinal epithelial cell membranes

Heat-stable enterotoxins are a family of toxin peptides that are produced by enterotoxigenic Escherichia coli and consist of 18 and 19 amino acid residues (Aimoto, S., Takao, T., Shimonishi, Y., Hara, S., Takeda, T., Takeda, Y., and Miwatani, T. (1982) Eur. J. Biochem. 129, 257-263). A synthetic fully toxic analog of the enterotoxin, Mpr5-STp(5-17), where Mpr is beta-mercaptopropionic acid and which consists of 13 amino acid residues from Cys5 to Cys17 in a heat-stable enterotoxin but is deaminated at its N terminus (Kubota, H., Hidaka, Y., Ozaki, H., Ito, H., Hirayama, T., Takeda, Y., and Shimonishi, Y. (1989) Biochem. Biophys. Res. Commun. 161, 229-235), has been crystalized from water, and its crystal structure has been solved by a direct method and refined by least square procedures to give an R factor of 0.089. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 21.010 (2) A, b = 27.621 (4) A, and c = 12.781 (1) A. The asymmetric unit of the crystals contains one peptide molecule with 13 water molecules. A right-hand spiral peptide backbone extends throughout the molecule. Three beta-turns are located along this spiral and fixed tightly by three intramolecular disulfide linkages. The actual structure predicts the biniding region on the enterotoxin to the receptor protein on the membrane of rat intestinal epithelial cells.     

Structural requirements for the intracellular subunit polymerization of the complement inhibitor C4b-binding protein

C4b-binding protein (C4BP), an important inhibitor of complement activation, has a unique spider-like shape. It is composed of six to seven identical alpha-chains with or without a single beta-chain, the chains being linked by disulfide bridges in their C-terminal parts. To elucidate the structural requirements for the assembly of the alpha-chains, recombinant C4BP was expressed in HEK 293 cells. The expressed C4BP was found to contain six disulfide-linked alpha-chains. Pulse-chase analysis demonstrated that the recombinant C4BP was rapidly synthesized in the cells and the polymerized C4BP appeared in the medium after 40 min. The alpha-chains were polymerized in the endoplasmic reticulum (ER) already after 5 min chase. The polymerization process was unaffected by blockage of the transport from the ER to the Golgi mediated by brefeldin A or low temperature (10 degrees C). The C-terminal part of the alpha-chain (57 amino acids), containing 2 cysteine residues and an amphiphatic alpha-helix region, was required for the polymerization. We constructed and expressed several mutants of C4BP that lacked the cysteine residues and/or were truncated at various positions in the C-terminal region. Gel filtration analysis of these variants demonstrated the whole alpha-helix region to be required for the formation of stable polymers of C4BP, which were further stabilized by the formation of disulfide bonds.     

Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments.

Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys-98 with Cys-125, and Cys-166 with Cys-200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18-33% residue identities) from microorganisms. However, one half-Cys (i.e., Cys-166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N-terminal part of the tomato pectinesterase, another in the C-terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to high-light a functional site in a midchain segment.     

Mechanism of insulin chain combination. Asymmetric roles of A-chain alpha-helices in disulfide pairing

The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal alpha-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the N-terminal alpha-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity. (1)H NMR studies of a representative analog lacking invariant side chains Ile(A2) and Val(A3) (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1-A5 segment in an otherwise native-like structure. That this and related partial folds retain efficient disulfide pairing suggests that the native N-terminal alpha-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed.     

Disulfide arrangement of human insulin-like growth factor I derived from yeast and plasma.

The disulfide arrangement of yeast derived human insulin-like growth factor I (yIGF-I) was determined using a combination of Staphylococcus aureus V8 protease mapping, fast-atom-bombardment mass spectrometry as well as amino acid sequence and composition analysis. Three disulfide bridges were found between the following cysteine residues: Cys6-Cys48, Cys47-Cys52 and Cys18-Cys61. IGF-I isolated from human plasma (pIGF-I) was found to have an identical disulfide configuration. A yeast-derived isomeric form of IGF-I (yisoIGF-I) exhibited an altered disulfide arrangement: Cys6-Cys47, Cys48-Cys52 and Cys18-Cys61. Radioreceptor analysis of pIGF-I and yIGF-I showed high specific activity, 20,000 U/mg. However, yisoIGF-I demonstrated a severely reduced ability to bind to the IGF-I receptor (19%) and was less potent in provoking a mitogenic response in Balb/C 3T3 fibroblasts (50% at doses 10-100 ng/ml). The data demonstrate the importance of correct disulfide arrangement in IGF-I for full biological activity.     

Characterization and DNA sequence of the b6w2 allotype of the rabbit immunoglobulin kappa 1 light chain (b locus)

The b6w2 allotype of the constant region of the rabbit immunoglobulin kappa 1 (k1) light chain (b locus) was discovered in wild populations from northern Spain. At the serological level, the b6w2 allotype is characterized by the presentation of all b6-specific epitopes, while an allotypic determinant which is shared between the nominal b5 and b6 allotypes is lacking. The DNA fragment encoding the b6w2 allotype was amplified by means of the polymerase chain reaction, and sequenced directly by dideoxy-DNA-sequencing. When compared with the sequence of the nominal b6 allele, the b6w2 sequence differs at eleven nucleotide positions (96.5% similarity). This variation corresponds to amino acid replacements at 1) the three positions C-terminal to the peptidyl junction with the variable region (amino acid positions 109–111);2) the four positions N-terminal to the interdomain disulfide bond (167–170); and 3) two positions in the vicinity of the interchain disulfide bond (190 and 210). The nature and distribution of the observed nucleotide substitutions strongly suggest a possible role of the extra interdomain disulfide bond in the unusual evolutionary dynamics of the rabbit K1 light chain.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number Z48308