Six Polymorphic Crystal Forms of α‐Cobratoxin

Abstract

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...