The three-dimensional crystal structure of variant-3 toxin from the scorpion Centruroides sculpturatus Ewing has been determined at 3 A resolution. Phases were obtained by use of K2PtCL4 and K2IrCl derivatives. The most prominent secondary structural features are two and a half turns of a-helix and a three-strand stretch of antiparallel a-sheet, which runs parallel to the a-helix. The helix is connected to the middle strand of the a-sheet by two disulfide bridges; a third disulfide bridge is located nearby. Several loops extend out of this dense core of secondary structure. The largest loop is joined to the COOH terminus of the molecule by the fourth disulfide bridge. The overall shape of the molecule resembles a right-hand fist: the a-helix runs along the knuckles of the fist; the a-sheet lies along the second and third joints of the fingers; the thumb is defined by two short loops that are composed of residues 16-21 and residues 41-46; the wrist corresponds to the COOHterminal stretch of residues 52-65 and a loop composed of residues 5-14; and the second joint of the little finger is near the NH2 terminus of the molecule. The a-carbon backbone displays a large flat surface that lies along the second joints of the fingers and the heel of the hand in the fist model. Several of the conserved residues in the scorpion neurotoxins are clustered on this surface, which may play a role in interactions of scorpion toxins with sodium channels of excitable membranes. Scorpion venoms contain sets of small basic proteins that are responsible for the neurotoxic activities of the venoms. These toxins display considerable variations in amino acid composition, but they generally consist of 60-70 amino acids and are crosslinked by four disulfide bridges. The partial or complete primary structures of about 25 toxins, from several different species of scorpions, have been determined (1). The amino acid sequences display a number of common features, including the same general locations of the eight cysteine residues, similar disulfide bridging patterns, and the location of several invariant or conserved residues. Thus, the available chemical data suggest that all of the scorpion toxins probably have similar threedimensional structures.The general effects of whole venoms or purified toxins injected in mammals are those expected from the massive release of neurotransmitters induced by depolarization of the nerve endings (2-4). Despite structural similarities among the various scorpion toxins, these proteins display wide variations in activity (5, 6), and it is not clear if all of the scorpion toxins follow the same general mechanism of action (7). Several of the toxins have been shown to prolong the action potentials of excitable membranes by blocking the inactivation of sodium channels (8-10), an effect similar to that produced by sea anemone toxins (11). However, the scorpion toxin and sea anemone toxin binding sites appear to be distinct from those occupied by otherThe publication costs of this article were defrayed in par...

1. The effect of various toxin fractions isolated by Watt et al. (1978) from the venom of the scorpion Centruroides sculpturatus Ewing on the Na currents of the node of Ranvier has been studied with the voltage clamp method. 2. The toxin fractions were applied externally. The most potent fractions were toxins III, IV and V which were effective in concentrations of 0.33-3.33 microgram/ml. The effect of toxins III and IV was quite different from that of toxin V. 3. In toxin III or IV - treated nodes a strong depolarizing pulse was followed by a transient shift of the negative resistance branch of the INa (E) curve to more negative potentials. The amount of shift varied between -10 and -60 mV. A 500 ms depolarizing pulse of small amplitude produced a slowly developing Na inward current which slowly decayed after the end of the pulse. Inactivation was incomplete, even with 500 ms pulses to 0 mV. 4. The transient shift of the INa (E) curve was not seen in nodes treated with toxin V. This toxin merely caused slow and incomplete Na inactivation. The effect of toxin IV was not suppressed by a four times higher concentration of toxin V, suggesting that the two toxins act on different receptors. 5. Toxin I acted like toxin IV but was about 10 times less potent. The effect of high concentrations of variants 1, 2, 3, 5, 6 resembled tha of toxin V. 6. All effects observed with toxin III or IV were also seen with the whole venom (cf. Cahalan 1975).

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It is evident from the data reviewed that scorpion toxins can be distinguished on the basis of three properties: their effects on Na currents, their specific binding to excitable membranes, and the effects of depolarization and pH on binding and on effect. Additional work with other scorpion toxins is required to establish the degree of correlation between the three properties for each class of toxin. Further investigations with this family of homologous proteins will undoubtedly contribute not only to our understanding of the toxins themselves but also to our understanding of the structure and function of the Na channel.

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The solution structure of the CsE-v3 neurotoxin from the venom of the North American scorpion Centruroides sculpturatus Ewing (CsE) has been determined by a hybrid refinement procedure that employed distance geometry and dynamical simulated annealing. Distance constraints deduced from the nuclear Overhauser effect spectroscopy data and torsion angle constraints deduced from the vicinal coupling constant data were used in the refinement procedure. A family of simulated annealing structures that showed no constraint violations was generated. The energy-minimized average structure exhibited root-mean-square deviations of 0.121 nm for the backbone and 0.182 nm for all atoms, with respect to this family. These studies confirm the previously qualitative NMR findings about the secondary structural features, viz. the presence of a short a-helix composed of residues 23-31 and an antiparallel P-sheet composed of the strands of residues 1-5, 45-50 and 36-42. A cluster of aromatic ring systems is located on one side of the protein. The solution and crystal structures have similar overall features, but show some minor differences.