A proton-magnetic-resonance study on the molecular conformation and structure-function relationship of a long neurotoxin, laticauda semifasciata III from Laticauda semifasciata.

The 300-MHz and 500-MHz NMR spectra of a long neurotoxin laticauda semifasciata III (LS III) from Laticauda semifasciata have ben analysed. Comparison with the NMR spectra of alpha-cobratoxin from Naja naja siamensis, a homologous long neurotoxin to laticauda semifasciata III, allowed the assignment of all the aromatic protein resonances to specific amino acid residues. All the methyl proton resonances have been assigned to specific types of amino acid residues. The pH dependences of the aromatic and methyl proton chemical shifts were analyzed by the non-linear least-square method to give the pKa values and protonation shifts. The interproton nuclear Overhauser effect enhancements were measured in order to elucidate the spatial proximity of the methyl-bearing residues and aromatic residues. On the basis of these NMR data and using the crystal structure of alpha-cobratoxin by Walkinshaw et al., more than half of the methyl proton resonances have been assigned to specific amino acid residues. A hydrophobic core comprising the first loop, the central loop and the tail part of the molecule has been defined. This hydrophobic core may be common to all long neurotoxins and may protect the three-stranded antiparallel pleated beta-sheet structure, thus making the backbone structure of long neurotoxins more rigid than that of short neurotoxins. The positively charged surface of laticauda semifasciata III, which is responsible for binding to the acetylcholine receptor protein, is confirmed as the concave surface formed by the central and the third loop. The arrangement of the amino acid residues on this surface is similar to that of all other neurotoxins. Accordingly, the slow on-off rates of association of long neurotoxins with receptor is considered to arise from the rigid backbone structure. A small conformation change is thought to be associated with binding to the receptor protein.