A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: comparison with long-chain alpha-neurotoxins and alpha-conotoxins.

Short-chain alpha-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short alpha-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain alpha-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30 A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III contact nAChR by their tips only in a 'surface-touch' manner. The toxin structure undergoes some changes during the final complex formation (for 1.45 rmsd in 15-25 ps according to AMBER'99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with alpha-cobratoxin and alpha-conotoxin. Among common features in binding of short- and long alpha-neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for alpha-conotoxin binding. Being in general very similar, the binding modes of short- and long alpha-neurotoxins differ in the ways of loop II entry into nAChR.

Polyclonal antibodies against native weak toxin Naja kaouthia discriminate native weak toxins and some other three-fingered toxins against their denaturated forms.

Polyclonal antibodies obtained by immunization of rabbits with native form of weak toxin (WTX) from cobra Naja kaouthia venom efficiently interacted with WTX and a weak toxin from Naja oxiana venom, but not so with their denaturated forms. These antibodies could also bind with lower affinity other groups of three-fingered toxins: long-chain alpha-neurotoxins, muscarinic toxins and cytotoxins, but practically did not bind short-chain alpha-neurotoxins. The efficiency of toxin-antibody interaction depends on the group (weak toxins, long or short alpha-neurotoxins, cytotoxins etc.) to which the toxin belongs, but not on species of snake from which the toxin originates. There is a correlation between the results obtained and phylogenetic analysis of the three-fingered toxins which revealed that WTX is very close to other weak toxins, relatively close to long alpha-neurotoxins, cytotoxins and muscarinic toxins, but is distant from the short alpha-neurotoxins.

Steered molecular dynamics simulations of cobra cytotoxin interaction with zwitterionic lipid bilayer: no penetration of loop tips into membranes.

Cobra cytotoxins, small proteins of three-fingered toxin family, unspecifically damage membranes in different cells and artificial vesicles. However, the molecular mechanism of this damage is not yet completely understood. We used steered molecular dynamics simulations to study the interaction of cardiotoxin A3 from Naja atra cobra venom with hydrated 1-palmitoyl-2-oleoyl-1-sn-3-phosphatidylcholine (POPC) bilayer. The studied system included one cytotoxin molecule, 64 lipid molecules (32 molecules in each monolayer) and 2500 water molecules. It was found that the toxin interacted with zwitterionic bilayer formed by POPC. During first nanosecond of simulation the toxin molecule was oriented toward membrane surface by loops' basement including cytotoxin regions Cys14-Asn19 and Cys38-Ser46. This orientation was stable enough and was not changed during next 6 ns of simulation. The obtained data suggest that cytotoxin molecule cannot penetrate into membrane composed of zwitterionic lipids without some auxiliary interaction.

Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits.

We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.