An nmr conformational analysis of a synthetic peptide Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from the New World Centruroides noxius 2 (Cn2) scorpion toxin: comparison of the structure with those of the Centruroides scorpion toxins

The solution structure of a synthetic peptide, Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from toxin 2 (Cn2) of the New World scorpion Centruroides noxius was determined using nmr and molecular dynamics calculations. The peptide has no significant secondary structure such as an alpha-helix or a beta-sheet, yet it has a fixed conformation for the first chain. The backbone secondary structure involving residues 6-12 in this peptide shows an excellent overlap with the structures of natural neurotoxins from Centruroides sculpturatus Ewing. Residues 6-9 form a distorted type I beta-turn and residues 10-12 form a gamma-turn. As residues 7-10 in the Centruroides toxins correspond to one of the regions of highest sequence variability, it may account for the species specificity and/or selectivity of toxic action. The conformation of this region evidently plays an important role in receptor recognition and in binding to the neutralizing monoclonal antibody BCF2 raised against the intact toxin.     

Mapping of an epitope recognized by a neutralizing monoclonal antibody specific to toxin Cn2 from the scorpion Centruroides noxius, using discontinuous synthetic peptides.

The Na+-channel-affecting toxin Cn2 represents the major and one of the most toxic components of the venom of the Mexican scorpion Centruroides noxius Hoffmann. A monoclonal antibody BCF2 raised against Cn2 has been shown previously to be able to neutralize the toxic effect of Cn2 and of the whole venom of C. noxius. In the present study the epitope was mapped to a surface region comprising the N- and C-terminal segments of Cn2, using continuous and discontinuous synthetic peptides, designed on the basis of the sequence and a three-dimensional model of Cn2. The study of peptides of varying length resulted in the identification of segments 5-14 and 56-65 containing residues essential for recognition by BCF2. The peptide (abbreviated SP7) with the highest affinity to BCF2 (IC50 = 5.1 microM) was a synthetic heterodimer comprising the amino acid sequence from position 3-15 (amidated) of Cn2, bridged by disulfide to peptide from position 54-66, acetylated and amidated. Similar affinity was found with peptide SP1 [heterodimer comprising residues 1-14 (amidated) of Cn2, bridged with synthetic peptide 52-66 (acetylated)]. SP1 and SP7 were used to induce anti-peptide antibodies in mouse and rabbit. Both peptides were highly immunogenic. The sera obtained were able to recognize Cn2 and to neutralize Cn2 in vitro. The most efficient protection (8.3 microgram Cn2 neutralized per mL of serum) was induced by rabbit anti-SP1 serum.     

NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity.

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.     

Amino acid sequence and immunological characterization with monoclonal antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann.

Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroides noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction II-9.2 (which contains toxins 2 and 3) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCF1 and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross-reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A-D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCF2 and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross-reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides suffusus suffusus = Centruroides infamatus infamatus, Centruroides limpidus tecomanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgG2a subclass, displayed a clear neutralizing activity in vivo.     

Isolation and primary structure of a potent toxin from the venom of the scorpion Centruroides sculpturatus Ewing.

A potent toxin has been purified from the venom of the scorpion Centruroides sculpturatus Ewing using the ion-exchange resin CM-Sepharose CL-6B at basic pH. The toxin, designated CsE M1, comprised 65 amino acid residues and its primary structure was established as: Lys-Glu-Gly-Tyr-Leu-Val-Asn-Ser-Tyr-Thr10-Gly-Cys-Lys-Tyr-Glu-Cys- Leu-Lys-Leu- Gly20-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Arg30-Gln-Gln-Tyr- Gly-Lys-Ser-Gly-Gly - Tyr-Cys40-Tyr-Ala-Phe-Ala-Cys-Trp-Cys-Thr-His-Leu50-Tyr-Glu- Gln-Ala-Val-Val-Trp - Pro-Leu-Pro60-Asn-Lys-Thr-Cys-Asn. CsE M1 is the most lethal protein to be identified in C. sculpturatus venom and the LD50 of the toxin, determined by subcutaneous injection into Swiss mice, is 87 micrograms/kg. CsE M1 shows strong structural similarity (92% positional identity) to the most potent beta-toxin, Css II, from the Mexican scorpion, Centruroides suffusus suffusus but is quite dissimilar to the previously characterized toxins with low potency isolated from C. sculpturatus Ewing.     

Three-dimensional structure of ATP:corrinoid adenosyltransferase from Salmonella typhimurium in its free state, complexed with MgATP, or complexed with hydroxycobalamin and MgATP

In Salmonella typhimurium, formation of the cobalt-carbon bond in the biosynthetic pathway for adenosylcobalamin is catalyzed by the product of the cobA gene which encodes a protein of 196 amino acid residues. This enzyme is an ATP:co(I)rrinoid adenosyltransferase which transfers an adenosyl moiety from MgATP to a broad range of co(I)rrinoid substrates that are believed to include cobinamide, its precursor cobyric acid and probably others as yet unidentified, and hydroxocobalamin. Three X-ray structures of CobA are reported here: its substrate-free form, a complex of CobA with MgATP, and a ternary complex of CobA with MgATP and hydroxycobalamin to 2.1, 1.8, and 2.1 A resolution, respectively. These structures show that the enzyme is a homodimer. In the apo structure, the polypeptide chain extends from Arg(28) to Lys(181) and consists of an alpha/beta structure built from a six-stranded parallel beta-sheet with strand order 324516. The topology of this fold is very similar to that seen in RecA protein, helicase domain, F(1)ATPase, and adenosylcobinamide kinase/adenosylcobinamide guanylyltransferase where a P-loop is located at the end of the first strand. Strikingly, the nucleotide in the MgATP.CobA complex binds to the P-loop of CobA in the opposite orientation compared to all the other nucleotide hydrolases. That is, the gamma-phosphate binds at the location normally occupied by the alpha-phosphate. The unusual orientation of the nucleotide arises because this enzyme transfers an adenosyl group rather than the gamma-phosphate. In the ternary complex, the binding site for hydroxycobalamin is located in a shallow bowl-shaped depression at the C-terminal end of the beta-sheet of one subunit; however, the active site is capped by the N-terminal helix from the symmetry-related subunit that now extends from Gln(7) to Ala(24). The lower ligand of cobalamin is well-ordered and interacts mostly with the N-terminal helix of the symmetry-related subunit. Interestingly, there are few interactions between the protein and the polar side chains of the corrin ring which accounts for the broad specificity of this enzyme. The corrin ring is oriented such that the cobalt atom is located approximately 6.1 A from C5' of the ribose and is beyond the range of nucleophilic attack. This suggests that a conformational change occurs in the ternary complex when Co(III) is reduced to Co(I).     

Directed evolution, phage display and combination of evolved mutants: a strategy to recover the neutralization properties of the scFv version of BCF2 a neutralizing monoclonal antibody specific to scorpion toxin Cn2

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.     

The crystal structure of tetanus toxin Hc fragment complexed with a synthetic GT1b analogue suggests cross-linking between ganglioside receptors and the toxin.

Tetanus toxin, a member of the family of Clostridial neurotoxins, is one of the most potent toxins known. The crystal structure of the complex of the COOH-terminal fragment of the heavy chain with an analogue of its ganglioside receptor, GT1b, provides the first direct identification and characterization of the ganglioside-binding sites. The ganglioside induces cross-linking by binding to two distinct sites on the Hc molecule. The structure sheds new light on the binding of Clostridial neurotoxins to receptors on neuronal cells and provides important information relevant to the design of anti-tetanus and anti-botulism therapeutic agents.     

Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins.

A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.     

kappa-Hefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function. Importance of the functional diad in potassium channel selectivity

An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, kappa-hefutoxin1 and kappa-hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that kappa-hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any beta-sheets. Based on the presence of the functional diad (Tyr(5)/Lys(19)) at a distance (6.0 +/- 1.0 A) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. kappa-Hefutoxin 1 not only blocks the voltage-gated K(+)-channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of kappa-hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.     

Cleavage of Type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the NH2-terminal portion of the smaller fragment released from both collagens.

Collagenase cleavage of human Type II and III collagens has been studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately five times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular weights and amino acid compositions of the products demonstrated that Type II and III molecules had been cleaved at the characteristic three-quarter, one-quarter locus, giving rise to a large fragment derived from the NH2-terminal portion of the molecule and a smaller fragment representing the COOH-terminal region. The amino acid sequence at the NH2-terminal portion of the smaller fragment derived from Type II collagen was determined to be Ile-Ala-Gly-Gln-Arg, and the corresponding region from Type III collagen was found to have the sequence Leu-Ala Gly-Leu-Arg. These sequences for alpha1(II) and alpha1(III) chains adjacent to the site of collagenase cleavage along with previous data for alpha1(I) and alpha2 chains indicate that the minimum specific sequence required for collagenase cleavage is Gly-Ile-Ala or Gly-Leu-Ala. Inspection of the available sequence data for collagen alpha chains indicates that the latter sequences are found in at least three additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase, however, are followed by a Gly-X-Hyp sequence. We suggest, then, that a minimum of five residues in collagen alpha chains COOH-terminal to the cleavage site comprise the substrate recognition site.     

A novel phospholipase A2, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor.

This paper describes the isolation and primary structure analysis of a new phospholipase A2 with platelet-aggregation-inhibiting activity from the venom of Bothrops jararaca. The protein, named BJ-PLA2, was isolated by means of ammonium sulfate precipitation and anion-exchange and reversed-phase chromatographies and behaved as a homogeneous single-chain protein on SDS-PAGE. Its amino acid sequence was determined by N-terminal sequencing and analysis of overlapped chemical and proteolytic fragments by automated Edman degradation and mass spectometry determination. BJ-PLA2 consists of 124 amino acid residues and has the structural features of snake venom class II phospholipases A2. Chemical modification with p-bromophenacylbromide caused complete loss of enzymatic activity and partially affected the platelet-aggregation-inhibiting activity of BJ-PLA2.     

Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex with 2'GMP.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.     

Interaction of paratropomyosin with beta-connectin and its 400-kilodalton fragment from chicken skeletal muscle as influenced by the calcium ion concentration.

The binding of paratropomyosin to beta-connectin, which has been suggested to interact at the A-I junction of a sarcomere, was confirmed by measuring the changes in turbidity of a mixture with changing NaCl concentration, pH and free calcium ions, and by morphological observation and a coprecipitation assay of the aggregates formed in the mixture. Paratropomyosin also bound to the 400-kDa fragment which is the N-terminal portion of beta-connectin and contains the A-I junction region. Moreover, the interaction of paratropomyosin with the 400-kDa fragment was enhanced by a calcium ion concentration from 10(-7) M to 10(-5) M and markedly suppressed above 10(-4) M calcium ions. We conclude that paratropomyosin probably binds to the 400-kDa fragment of beta-connectin in the A-I junction region in living and pre-rigor skeletal muscle. In postmortem skeletal muscle paratropomyosin may be released from the 400-kDa portion of the connectin filament by increased calcium ion concentration and translocated on to thin filaments to induce meat tenderization.     

The three-dimensional structures of nicotinate mononucleotide:5,6- dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium complexed with 5,6-dimethybenzimidazole and its reaction products determined to 1.9 A resolution

Nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium plays a central role in the synthesis of alpha-ribazole, which is a key component of the lower ligand of cobalamin. Two X-ray structures of CobT are reported here at 1.9 A resolution. First, a complex of CobT with 5,6-dimethylbenzimidazole, and second, a complex of CobT with its reaction products, nicotinate and alpha-ribazole-5'-phosphate. CobT was cocrystallized with 5,6-dimethylbenzimidazole (DMB) in the space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 A, b = 90.2 A, and c = 47.5 A and one protomer per asymmetric unit. Subsequently, the crystals containing DMB were soaked in nicotinate mononucleotide whereupon the physiological reaction occurred in the crystal lattice to yield nicotinate and alpha-ribazole-5'-phosphate. These studies show that CobT is a dimer where each subunit consists of two domains. The large domain is dominated by a parallel six-stranded beta-sheet with connecting alpha-helices that exhibit the topology of a Rossmann fold. The small domain is made from components of the N- and C-terminal sections of the polypeptide chain and contains a three-helix bundle. The fold of CobT is unrelated to the type I and II phosphoribosylpyrophosphate dependent transferases and does not appear to be related to any other protein whose structure is known. The enzyme active site is located in a large cavity formed by the loops at the C-terminal ends of the beta-strands and the small domain of the neighboring subunit. DMB binds in a hydrophobic pocket created in part by the neighboring small domain. This is consistent with the broad specificity of this enzyme for aromatic substrates [Trzebiatowski, J. R., Escalante-Semerena (1997) J. Biol. Chem. 272, 17662-17667]. The binding site for DMB suggests that Glu317 is the catalytic base required for the reaction. The remainder of the cavity binds the nicotinate and ribose-5'-phosphate moieties, which are nestled within the loops at the ends of the beta-strands. Interestingly, the orientation of the substrate and products are opposite from that expected for a Rossmann fold.