Crystalline erabutoxin c

Preston, H. S.; Kay, Jack; Sato, Atsushi; Low, Barbara W.; Tamiya, Nanako

doi:10.1016/0041-0101(75)90133-6

Cited by 4 publications

(5 citation statements)

References 5 publications

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“…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 The molecule is an elongated concave disk or shallow saucer with foot. Two long chain regions that consist of well-defined loops were recognized in the 2.9 A electron density map (16).…”

mentioning

confidence: 99%

“…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, b = 46.69 A, and c = 21.43 A; there is one molecule per asymmetric unit.…”

mentioning

confidence: 99%

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

Low¹,

Preston²,

Sato³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

225

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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,...

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mentioning

confidence: 99%

“…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, b = 46.69 A, and c = 21.43 A; there is one molecule per asymmetric unit.…”

mentioning

confidence: 99%

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

Low¹,

Preston²,

Sato³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

225

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“…Using experimental conditions identical to those described above, we found the same crystal form for Ea and Eb [5]. This crystal form was originally described by Preston et al [6] for the wild-types Ec and Eb. The unit cell parameters of erabutoxins crystallized with SCN-are indicated in Table 1.…”

Section: Crystallizationmentioning

confidence: 49%

“…These crystallization conditions lead to a dimeric form, which was originally reported by Preston et al, for EC and Eb [6]. X-ray diffraction data were recorded and the 3D structure of Ea, was solved at 2.0 A resolution starting from the 3D structure of Eb crystallized under the same conditions [5].…”

Section: In~~u~tionmentioning

confidence: 99%

Three‐dimensional crystal structure of recombinant erabutoxin a at 2.0 Å resolution

Arnoux¹,

Ménez

Drevet

et al. 1994

FEBS Letters

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Recombinant erabutoxin a (Ea ) has been crystallized by vapour diffusion in hanging drops. The crystals belong to space group P2,2i2, with cell dimensions a = 55.8 A. b = 53.4 A. c = 40.8 A. Diffraction data have been recorded on a FAST detector un to 2.0 A. The atomic crystal structure of Ea, has been determined by initial refinement of the structure of the isotoxin erabutoxin b (Eb) the cry&Is of which were grown under identical conditions. The R-factor was 23% at 2.0 A resolution. The secondary and tertiary structures of Ea, are shown to be identical with that of wild-type Eb, within the experimental error.

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“…The X-ray crystal structures of the 'short' erabutoxin b [2,3] and of the 'long' a-cobratoxin [4] provided insight into this binding mechanism. Of the short toxins, cobrotoxin [5] and erabutoxins a, b and c [6,7] have been crystallised. Despite efforts in a number of laboratories, attempts at crystallising any of the long toxins were unsuccessful, except for the rx-cobratoxin reported here and for a-bungarotoxin, the 'long' neurotoxin of Bungarus multicinctus, which was crystallised and subjected to X-ray crystal analysis (R. M. Stroud, personal communication).…”

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confidence: 99%

Six Polymorphic Crystal Forms of α‐Cobratoxin

Walkinshaw

1981

European Journal of Biochemistry

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Six different crystal forms of the long neurotoxin a-cobratoxin isolated from the venom of Nuja nuja siamerisis have been obtained and the conditions for their crystallisation are described. Heavy atom derivatives for two of these forms have been prepared and have proven suitable for high-resolution X-ray studies and, with one of them, a 0.28-nm resolution structure has been determined. All but one of the crystal forms were grown between p H 2 and pH 3. Two novel suggestions are made which may be applicable to other protein crystallisation studies. One involves cocrystallisation with mercurous iodide and the other describes a two-step microdialysis procedure, first against salt solution, followed by dialysis against poly(ethy1ene glycol).The neurotoxin a-cobratoxin consists of 71 amino acids and has a molecular weight of 7803. Over 60 post-synaptic neurotoxins isolated from snake venom have been sequenced. They are classified into two groups [l]: the 'short' toxins consist of 60-62 amino acids and have four disulphide bridges while the 'long' neurotoxin family have 66 -74 amino acids with five disulphide bridges. Both long and short neurotoxins bind strongly to and inhibit the acetylcholine receptor function. The X-ray crystal structures of the 'short' erabutoxin b [2,3] and of the 'long' a-cobratoxin [4] provided insight into this binding mechanism. Of the short toxins, cobrotoxin [5] and erabutoxins a, b and c [6,7] have been crystallised. Despite efforts in a number of laboratories, attempts at crystallising any of the long toxins were unsuccessful, except for the rx-cobratoxin reported here and for a-bungarotoxin, the 'long' neurotoxin of Bungarus multicinctus, which was crystallised and subjected to X-ray crystal analysis (R. M. Stroud, personal communication).Various parameters including pH, protein concentration, ionic strength, nature of precipitating agent and temperature were varied systematically in order to determine optimal conditions for crystal growth. The eventual success in growing six different crystal forms of the long neurotoxin x-cobratoxin, two of which were suitable for a high-resolution X-ray study, provides some general additional information about the techniques of growing protein crystals. EXPERIMENTAL PROCEDURE n-Cobratoxin from Naju nuja siumensis ( N . n. kaoutlzia) venom (Miami Serpentarium Florida), purified by ion-exchange chromatography on phosphocellulose, carboxymethylcellulose and Bio-Rex 70 columns, was kindly provided by Professor A. Maelicke. The pH of the crystallising media was monitored in steps of 0.3 from pH 1.5 using standard buffers [8]. Sodium azide (0.005 M) was added to the buffers to prevent bacterial growth. Crystals were obtained by employing two techniques of isothermal distillation and microdialysis.For isothermal distillation (vapour diffusion), aliquots of between 10 pl and 50 p1 protein solution were pipetted into siliconised glass phials (1-cm deep x 0.5-cm diameter) and placed in sealed petri dishes containing the buffered precipitating medium.For micro...

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Crystalline erabutoxin c

Cited by 4 publications

References 5 publications

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

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

Three‐dimensional crystal structure of recombinant erabutoxin a at 2.0 Å resolution

Six Polymorphic Crystal Forms of α‐Cobratoxin

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