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In peptide—protein and protein—protein interactions the size of the buried surface area ranges from 400
Å 2 to 1400 Å 2 [66]. In the potassium channel blocker charybdotoxin, 5–8 residues with surface areas of
530–850 Å 2 were found to be essential for binding, depending on the type of potassium channel
investigated [67,68]. In the calcium channel blocker ω-conotoxin GVIA, an alanine scan identified only
two residues, Lys2 and Tyr13, that were important for activity [69]. It is likely, however, that the
number of residues contributing to the binding surface is greater than this, particularly given the high
affinity of this toxin for its receptor. If we consider a larger ligand such as human growth hormone, eight
of the 31 residues in the growth hormone-receptor interface contribute 85% of the binding energy and
more than half make no significant contribution to the affinity [70]. A similar result was found for a
growth hormone-monoclonal antibody complex, where only five residues were critical for binding [71].
In fact, the example of growth hormone may be more relevant to the anthopleurins than those of
charybdotoxin and conotoxin, which function simply as ion channel blockers. Thus, we expect the
essential residues in the anthopleurins to number between five and ten, a total somewhat less than
previously anticipated [24].
If we ignore Asp9 and assume that only one of Arg12 or Arg14 and one of Lys48 or Lys49 are
necessary, then it is likely that the residues highlighted in Figure 6 make a significant contribution to the
sodium channel binding surface of the anthopleurins. They span a larger area on the surface than the
essential residues in charybdotoxin, but it is important to note that the conformation of the Arg14 loop in
solution is not fixed and that conformations in which the Arg14 side chain is closer to the region around
Asp7 could be more representative of the sodium-channel-bound structure. For example, the distance
between the Arg14 guanidino group and the Asp7 carboxylate varies from 13 to 26 Å over the family of
structures of AP-A and 10 to 22 Å in AP-B.
By analogy with other protein—protein interactions, it is likely that the sodium channel binding surface
of the anthopleurins will include side chains that mutagenesis studies will not identify as having a
significant role in binding or activity. As indicated above, alanine scanning of residues in the human
growth hormone—receptor interface indicated that less than a quarter of the contact residues provided
most of the binding energy [70]. Thus, we believe that the residues identified above will contribute to
the sodium channel binding surface of the anthopleurins by virtue of their location on the protein surface
in the immediate vicinity of residues, which clearly are important for activity. Nevertheless, some of
them may make only modest contributions to binding affinity and could be altered without destroying
activity. Other residues, as yet unidentified, may also make significant contributions. By analogy with
other protein—protein interactions characterized to date, it is reasonable to anticipate that some of these
residues will be hydrophobic, in contrast to the charged
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