Conformational Studies of Neurotoxin II from Naja naja oxiana

Abstract

After treatment of neurotoxin 11, a component part of the venom of the Middle Asian cobra Naja naju oxiana, with acetoxysuccinimide all five possible e-acetylated-lysyl derivatives were obtained and the position of the label was established. Trifluoroacetylation of both the derivatives and the parent toxin yielded, respectively, the five acetyl-penta(trifluoroacety1)-neurotoxins I1 and the hexa(trifluoroacety1)-neurotoxin 11, which were studied by circular dichroism (CD), 'H and I9F nuclear magnetic resonance (NMR) spectroscopy. The availability of this series of compounds made possible assignment of all six fluorine signals (from the N-terminal and the five &-amino groups) in the hexa(trifluoroacety1)-neurotoxin 11 NMR spectra and disclosure of the proximity of the Lys-26 and Lys-46 trifluoroacetyl groups. The pH dependence of the "F NMR signals was determined and the pK values of the groups affecting the signal chemical shifts were calculated by a computer iterative program. In order to ascertain the relative accessibility of the lysyl side chains, the change in halfwidths of the hexatrifluoroacetylated neurotoxin I1 I9F signals, with addition of varying amounts of an iminoxyl spin probe, was determined. The data obtained are compared with the X-ray data on sea snake neurotoxins and the significance of the side chain interactions observed in solution is discussed.Among the various components of snake venom, the neurotoxins are endowed not only with high potency but with an exceptional specificity of action. The most extensively studied members of this group, the postsynaptic neurotoxins, block transmission of the nerve impulse by binding to the acetylcholine receptor in the membrane [l -41. The present work is a continuation of our spectroscopic studies of postsynaptic neurotoxins [5 -71 aiming at their dynamic molecular structure in solution, this being of crucial importance for understanding toxin-receptor interactions. Up to now the most informative results have been obtained from the 'H NMR study of neurotoxin 11, the primary structure of which is given in Fig.1. In the above-mentioned work, chemical modification was used as an aid in the spectral assignments; however, in the present study it is used as an equal partner of NMR, considerably broadening its scope.We prepared a neurotoxin I1 derivative whose amino groups were all trifluoroacetylated and conAbbreviations. CD, circular dichroism, NMR, nuclear magnetic resonance; ESR, electron spin resonance; spin probe, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl.--~ ducted the 19F NMR study of this compound, the signals of which were assigned with the aid of a series of selectively acetylated-trifluoroacetylated derivatives. As a result, intramolecular interactions and the extent of solvent exposure of trifluoroacetyl groups were discovered; this, together with the earlier data [6,7], yields a better understanding of the folding of the neurotoxin backbone.,.-.

EPR And fluorescence study of interaction of Naja naja oxiana neurotoxin II and its derivatives with acetylcholine receptor protein from Torpedo marmorata

“…The neurotoxin binding site is apparently considerably extended and the neurotoxin-receptor interaction, as was supposed [2] is of a multipoint character involving at least the 3 loops A, B and C confined with disulfide bonds (fig.4). The present data support in particular our earlier supposition that the loops B and C are involved in binding [4]. obtained with other neurotoxins [2,29,30].…”

“…Compounds I-III, VI and VIII-XII (see table 1) were obtained according to [4,14,15 1, the preparation of compounds IV, V and VII (synthesized via spin-labeled N-hydroxysuccinimide ester) will be published elsewhere. AchR from the electric organ of Torpedo marmarata was isolated by the procedure in [ 16 1.…”

“…As the solution structure of neurotoxin II might be essentially similar [4,15] to the crystal structure of erabutoxin b [23,31,32] we have used the crystal structure to illustrate the binding of neurotoxins to AchR (fig.4). The figure shows the association of one neurotoxin molecule.…”

“…We attacked this problem by using EPR and fluorescence spectroscopy for monitoring the binding of selectively spin-and fluorescence-labeled derivatives of the short (61 residues) neurotoxin II of N@z najz oxiana with AchR from the electric organ of Torpedo marmorata. The respective reporter groups were attached to lysine e-amino groups, since upon acylation of the latter the spatial structure and biological activity are essentially preserved [4].…”

The 1H Nuclear-Magnetic-Resonance Spectra and Spatial Structure of the Naja mossambica mossambica Neurotoxin III

“…The existence of a common conformational type for the short-chain neurotoxins has also been confirmed by NMR studies on the members of the homologous series including neurotoxin I1 of N . n. oxiuna [10,11,[13][14][15][16][17][18], a-toxin of Naja nigricollis [12], cobrotoxin of Naja nuja atra [22,23] …”