Refined structure in solution of the sea anemone neurotoxin ShI

A high resolution structure in solution has been determined for the polypeptide neurotoxin I, from the sea anemone Stichodactyla helianthus, using NMR data, distance geometry calculations, and refinement by back- calculation of two-dimensional nuclear Overhauser enhancement (NOE) spectra. A set of 913 distance constraints derived from NOEs was used, together with a large set of lower distance bounds based on the absence of NOEs in the spectrum. Eight published structures for neurotoxin I were refined independently to give structures which agree better with the experimental data, as reflected in reduced R factors calculated over well resolved cross- peaks of the two-dimensional NOE spectra and a lower total volume of peaks in back-calculated spectra that are absent from experimental spectra. The refined structures are also more precisely defined, with mean pairwise root mean square differences over backbone heavy atoms of 0.62 Å for well defined residues and 1.14 Å for all residues, compared with previous values of 1.09 and 2.41 Å, respectively. The consensus constraint set from the 8 refined structures was also used to generate 12 new structures, with corresponding root mean square differences of 0.76 and 1.26 Å. In all 20 structures the loop linking the first and second strands of the β-sheet is considerably better defined than before. A type I β-turn encompassing the functionally important residues Asp⁶, Asp⁷, and Glu⁸ is evident in the refined structures.