Three dimensional structure of erabutoxin b neurotoxic protein: inhibitor of acetylcholine receptor.

Abstract

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 monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short‐chain variants

“…Amino acid residues incorporated in the epitope recognized by Mcr2-3. The spatial structure is based on X-ray data of erabutoxin b [23,24] according to Kimball et al [22]. The three underlined residues at position 27, 29 and 47 are common to both the epitope and the AcChR-binding site.…”

Refinement at 1.4 Å resolution of a model of erabutoxin b: treatment of ordered solvent and discrete disorder

“…The threedimensional structure of erabutoxin b was reported previously from analyses at lower resolution (Low, Preston, Sato, Rosen, Searl, Rudko & Richardson, 1976;Kimball, Sato, Richardson, Rosen & Low, 1979) as were the results of an earlier stage of the crystallographic refinement at 1.4 A resolution (Bourne, Sato, Corfield, Rosen, Birken & Low, 1985;Low & Corfield, 1986). The earlier refinement provided starting parameters for the work reported here, corrected errors in the amino-acid sequence of erabutoxin b, and established the identity of erabutoxin b and neurotoxin b, toxins from the venom of L. semifasciata from different regions of the Pacific.…”

“…The diffraction data employed in the initial stages of this study were those used earlier (Low et al, 1976;Kimball et al, 1979) and in the phases 0 through IV of refinement that are described in the preliminary report (Bourne et al, 1985). This data set did not include estimated standard deviations for structure amplitudes.…”

Interaction of modified neurotoxins fromNaja nigricollis with the nicotinic acetylcholine receptor fromTorpedo marmorata A Raman spectroscopy study

“…Moreover', the toxin is not denatured as indicated by the high remaining toxicity and by its unchanged affinity towards the M~l antibody [7]. This value of the v(NO2) frequency could be related to • ~e fact that Tyr zs of the homologous erabutoxin does not appear completely buried, upon examination of the X-ray structure, albeit inaccessible [25]. Bat, if Tyr -~5 was involved in direct binding to a side-chain of the receptor by means of a phenol H-bond, this frequency would change.…”