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location). Another is that both the backbone and the side chains of residues 8– 16 are less well defined
than the bulk of the structure due to a lack of medium- and long-range NMR restraints between residues
in this loop and the rest of the molecule, as illustrated in Figure 5. These problems are exacerbated in the
case of AP-A and AP-B by the presence of multiple conformers in solution, one cause of which is cistrans
isomerism about the Gly40-Pro41 peptide bond [52]. The additional peak overlap caused by these
conformers limited the number of NOE restraints that could be obtained from the spectra. Finally, the
structures available at present are for the free ligand and we have no information on how much these
structures might change upon binding to the sodium channel.
One way of addressing the issue of a lack of precision in the locations of functionally important side
chains is to determine the range of conformational space available to them in different ligands. To this
end, we undertook a detailed comparison of the structures of AP-A and AP-B in solution [47]. This
proved to be a useful exercise both in terms of defining the positions of side chains known to be
important for cardiotonic activity and identifying neighboring residues which might also be involved
[47].
Models have been described in the literature for AP-B [53] and Bunodosoma granulifera toxins II (Bg
II) [26]. The AP-B model was derived from the structure of Sh I using energy minimization and the Bg
II model from that of BDS I using energy minimization and 10 ps of dynamics. In both cases the
calculations appear to have been carried out for the molecule in vacuo without the use of a distancedependent
dielectric, under which conditions the positions of the charged side chains on the surface are
likely to be distorted. Visual comparison of the model of AP-B [53] with the experimentally determined
solution structure [47] indicates significant differences in side-chain orientations.
IV. Residues Essential For Cardiotonic Activity
Information about which residues are essential for the cardiac stimulatory activity of the sea anemone
toxins has been obtained from selective chemical modification and proteolysis studies, comparisons
among naturally occurring sequences, and, most recently, site-directed mutagenesis. Although there are
some discrepancies among the inferences drawn from different studies and different techniques, a
consensus is emerging regarding the location of the cardioactive pharmacophore. Ideally, only effects on
cardiac tissue should be considered, but doing so would exclude some useful data on activity against
mammalian nerve preparations. However, data obtained on the Type 2 toxins or on the activity of Type
1 toxins on nonmammalian tissues will not be discussed in detail.
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