alpha-cobratoxin and erabutoxin-b

The review will critically assess the information available on the conformation of homologous neurotoxins and cytotoxins isolated from Elapid snakes. Particular attention will be given to the dynamics of the molecules in solution because there is the possibility that defined intramolecular rearrangements are involved at the sites of action. Such properties will be then reconciled with the known X-ray crystallographic and sequence data in order to derive likely structure-activity relationships.

Topics: Amino Acid Sequence; Animals; Bungarotoxins; Chemical Phenomena; Chemistry; Chemistry, Physical; Circular Dichroism; Cobra Neurotoxin Proteins; Cytotoxins; Drug Stability; Elapid Venoms; Erabutoxins; Hot Temperature; Magnetic Resonance Spectroscopy; Molecular Conformation; Neurotoxins; Receptors, Cholinergic; Solutions; Solvents; Structure-Activity Relationship; X-Ray Diffraction

Topics: Amino Acid Sequence; Chemical Phenomena; Chemistry; Cobra Neurotoxin Proteins; Disulfides; Elapid Venoms; Erabutoxins; Ethylmercury Compounds; Histidine; Kinetics; Magnetic Resonance Spectroscopy; Protein Conformation; Receptors, Cholinergic; Structure-Activity Relationship; Tyrosine; X-Ray Diffraction

Snake curaremimetic toxins are short all-beta proteins, containing several disulfide bonds which largely contribute to their stability. The four disulfides present in snake toxins make a "disulfide beta-cross"-fold that was suggested to be a good protein folding template. Previous studies on the refolding of snake toxins (Ménez, A. et al. (1980) Biochemistry 19, 4166-4172) showed that this set of natural homologous proteins displays different rates of refolding. These studies suggested that the observed different rates could be correlated to the length of turn 2, one out of five turns present in the toxins structure and close to the "disulfide beta-cross". To demonstrate this hypothesis, we studied the refolding pathways and kinetics of two natural isotoxins, toxin alpha (Naja nigricollis) and erabutoxin b (Laticauda semifasciata), and two synthetic homologues, the alpha mutants, alpha60 and alpha62. These mutants were designed to probe the peculiar role of the turn 2 on the refolding process by deletion or insertion of one residue in the turn length that reproduced the natural heterogeneity at that locus. The refolding was studied by electrospray mass spectrometry (ESMS) time-course analysis. This analysis permitted both the identification and quantitation of the population of intermediates present during the process. All toxins were shown to share the same sequential scheme for disulfide bond formation despite large differences in their refolding rates. The results presented here demonstrate definitely that no residues except those forming turn 2 accounted for the observed differences in the refolding rate of toxins.

Topics: Alkylation; Amino Acid Sequence; Amino Acid Substitution; Animals; Cobra Neurotoxin Proteins; Erabutoxins; Mass Spectrometry; Molecular Sequence Data; Mutation; Peptide Mapping; Protein Folding; Protein Structure, Secondary

The relationship between beta-sheet secondary structure and intrinsic tryptophan fluorescence parameters of erabutoxin b, alpha-cobratoxin, and alpha-bungarotoxin were examined. Nuclear magnetic resonance and x-ray crystallography have shown that these neurotoxins have comparable beta-sheet, beta-turn, and random coil secondary structures. Each toxin contains a single tryptophan (Trp) residue within its beta-sheet. The time-resolved fluorescence properties of native erabutoxin b and alpha-cobratoxin are best described by triple exponential decay kinetics, whereas native alpha-bungarotoxin exhibits more than four lifetimes. The disulphide bonds of each toxin were reduced to facilitate carboxymethylation and amidocarboxymethylation. The two different toxin derivatives of all three neurotoxins displayed triple exponential decay kinetics and were completely denatured as evidenced by circular dichroism (random coil). The concentration (c) values of the three fluorescence decay times (time-resolved fluorescence spectroscopy (TRFS)) were dramatically different from those of the native toxins. Each neurotoxin, treated with different concentrations of guanidinium hydrochloride (GuHCl), was studied both by circular dichroism and TRFS. Disappearance of the beta-sheet secondary structural features with increasing concentrations of GuHCl was accompanied by a shift in the relative contribution (c value) of each fluorescence decay time (TRFS). It was found that certain disulphide residues confer added stability to the beta-sheet secondary structure of these neurotoxins and that the center of the beta-sheet is last to unfold. These titrations show that Trp can be used as a very localized probe of secondary structure.

Topics: Animals; Biophysical Phenomena; Biophysics; Bungarotoxins; Circular Dichroism; Cobra Neurotoxin Proteins; Erabutoxins; Guanidine; Guanidines; In Vitro Techniques; Models, Molecular; Neurotoxins; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Spectrometry, Fluorescence; Tryptophan

Structural features associated with the ability of a monoclonal antibody (mAb) to discriminate between protein variants are identified and engineered. The variants are the curaremimetic toxin alpha from Naja nigricollis and erabutoxin a or b from Laticauda semifasciata, which differ from each other by 16 substitutions and one insertion. The neutralizing mAb M alpha 1 recognizes with high affinity a topographical epitope on the surface of toxin alpha, but fails to recognize the erabutoxins although they possess most of the residues forming the presumed epitope. Examinations of the toxin alpha and erabutoxin 3-D structures and molecular dynamics simulations reveal several differences between the variants. In particular, the region involving the beta-turn 17-24 is organized differently. Analysis of the differences found in this region suggest that the insertion (or deletion) at position 18 of the variant amino acid sequences is particularly important in determining the differential cross-reactivity. To test this proposal, residue 18 was deleted in one erabutoxin using site-directed mutagenesis, and the biological properties of the resulting mutant were examined. We found that full antigenicity was restored in the previously unrecognized variant. The implications of this finding are discussed.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antigen-Antibody Reactions; Cholinergic Antagonists; Cobra Neurotoxin Proteins; Computer Simulation; Cross Reactions; Epitopes; Erabutoxins; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Engineering; Recombinant Fusion Proteins; Sequence Deletion

The solution conformation of toxin alpha from Naja nigricollis (61 amino acids and four disulfides), a snake toxin which specifically blocks the activity of the nicotinic acetylcholine receptor (AcChoR), has been determined using nuclear magnetic resonance spectroscopy and molecular modeling. The solution structures were calculated using 409 distance and 73 dihedral angle restraints. The average atomic rms deviation between the eight refined structures and the mean structure is approximately 0.5 A for the backbone atoms. The overall folding of toxin alpha consists of three major loops which are stabilized by three disulfide bridges and one short C terminal loop stabilized by a fourth disulfide bridge. All the disulfides are grouped in the same region of the molecule, forming a highly constrained structure from which the loops protrude. As predicted, this structure appears to be very similar to the 1.4-A resolution crystal structure of another snake neurotoxin, namely, erabutoxin b from Laticauda semifasciata. The atomic rms deviation for the backbone atoms between the solution and crystal structures is approximately 1.7 A. The minor differences which are observed between the two structures are partly related to the deletion of one residue from the chain of toxin alpha. It is notable that, although the two toxins differ from each other by 16 amino acid substitutions, their side chains have an essentially similar spatial organization. However, most of the side chains which constitute the presumed AcChoR binding site for the curaremimetic toxins are poorly resolved in toxin alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Cobra Neurotoxin Proteins; Erabutoxins; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Protein Folding; Protein Structure, Secondary; Protons