The three-dimensional structure of erabutoxin b, a neurotoxin in the venom of the sea snake Laticauda semifasciata, has been determined from a 2.75 A resolution electron density map. Erabutoxin b is one of a family of snake venom neurotoxins, all low-molecular-weight proteins, which block neuromuscular transmission at the postsynaptic membrane. They specifically inhibit the acetylcholine receptor.The molecular shape is that of a shallow elongated saucer with a footed stand formed by the six-membered ring at the COOH-terminal end. The central core of the molecule is an assembly of four disulfide bridges. Three long chain loops emerge as broad fronds from the core region. Approximately 40% of the main chain is organized into a twisted antiparaliel -pleated sheet of five short strands. In 28 snake venom neurotoxins of established sequence which inhibit the acetylcholine receptor, the four disulfide bridges and seven other residues remain invariant. Three substitution positions conserve residue type. In one wing of the molecule, there is a broad shallow depression which may characterize the reactive site. It is populated by the seven invariant residues and two of the three type conserved residues. This region is "anchored" on the undersurface of the molecule by the hydroxyl group of Ser-9, the remaining conservatively substituted residue.Erabutoxin b is a protein neurotoxin of low molecular weight from the venom of the sea snake Laticauda semifasciata which blocks neuromuscular transmission at the postsynaptic membrane (1), in a nondepolarizing curare-like mode. The toxin binds at the cholinergic receptor site (2). Neurotoxins from sea and land snakes in the families Hydrophfidae and Elapidae show close structural homology (3) and a common mode of action (4-7). These neurotoxins specifically inhibit the acetylcholine membrane receptor protein (8,9).Three neurotoxins of Laticauda semifasciata are basic (isoelectric point >pH 9.2) single-chain proteins with 62 amino-acid residues and four disulfide bridges (10-12). Erabutoxins a and c are both single-substituted variants of erabutoxin b. The sequences of seven sea snake toxins, all with 60-62 residues, show multiple sequence substitutions and a dipeptide deletion in two toxins (3). When both sea and land snake neurotoxins are considered, the common mode of action and structural homology of 28 toxins is conserved through a broader range of sequence changes which include substitutions and deletions as well as insertions and COOH-terminal extensions. There remain the four invariant disulfide bridges as well as seven other invariant residues and three substitution positions which conserve residue type (see Fig. 1). We use the erabutoxin b sequence enumeration throughout. Preliminary x-ray diffraction studies (13,14) of erabutoxin b and the toxic erabutoxin b iodinated at His-26 (15) were reported and some features of earlier electron density maps at 5 Aand 2.9 A described (16). In the orthorhombic crystal, space group P212121, employed in this study, a = 50.01 A,...

A study has been made, following high-resolution refinement at 0.14 nm, of the structure of erabutoxin b, prototype postsynaptic neurotoxin from snake venom. The detailed patterns of intramolecular van der Waal's interactions have been determined. From information, hitherto unavailable, about atomic temperature parameters, the relative mobilities in different regions of the molecule have been estimated. A detailed model of structure/function relationships in these neurotoxins, whch bind to the acetylcholine receptor, has thus been established: the probable dynamic mode of toxin-receptor binding is described. The model identifies, and the binding mode depends on a unique structural feature of these protein toxins: the hydrophobic 'Trp' cleft. Chargecharge interactions are implicated in initial toxin orientation on the receptor surface. Possible reactive-site extension in short-chain toxins is described. Modifications in binding mode of long-chain toxins are considered. The relative mobilities of antigenic site residues are discussed.The postsynaptic neurotoxins from snake venom have been widely employed as probes of nicotinic acetylcholine receptor (AChR) function. Determination of the three-dimensional structure [l] of one of these protein toxins, erabutoxin b (Eb), led to early identification and characterization of the toxin reactive site [l -31, that interactive surface domain which binds to the receptor. Subsequent high-resolution refinement at 0.14 nm of this toxin structure [4] provides a more detailed picture and clearer understanding of the probable stereochemical aspects and dynamic features of toxin-receptor binding interactions.These neurotoxins bind to receptor competitively with acetylcholine (ACh) [5]. They produce a non-depolarising neuromuscular block. Extensive sequence homologies [2,6,7] in this large class of small single-chain proteins are expressed not only in the numbers of invariant (I) and type-conserved (T-C) residues, but also by their distribution along invariant length segments of peptide chain. The homologies link both series of toxins: short-chain, with 60-62 residues and four invariant disulfide bridges, and long-chain with, usually, 71 -74 residues and a fifth additional disulfide bridge. Initial characterization [2] of the toxin reactive site was therefore based on consideration of the stereochemical distribution of conserved residues as found in the prototype erabutoxin bCorrespondence to B. W. Low,

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Homo-17a-oxa-l,4-androstandiene-3,17-dione, 2.00 g., was dissolved in 60 ml. of pyridine and hydrogen sulfide was bubbled into the solution for two hours. Then 2 drops of piperidine was added and the reaction was allowed to stand for 3 days. After this time 200 ml. of water was added and the solution was extracted three times with ether. A solid formed in the aqueous layer and was separated by filtration.It was crystallized from ethyl acetate to give 0.34 g. of la,-5a-epidithio-17a-oxa-D-homoandrostane-3,17-dione, m.p. 232-233°dec., red melt, [«]d -140 ± 2°,