*3.2. κ-conotoxin PVIIA*

The drosophila Shaker (Shaker) channel is the first voltage-gated potassium channel to be cloned, and therefore it is often used as a role model in investigation of the structure and function of potassium channels. Similarly, *κ*-PVIIA is the first conotoxin found to block Shaker [75], and thus it has attracted a great deal of attention. The binding mode of *κ*-PVIIA to Shaker was studied via mutagenesis experiments [76,77], and it has also been used in many functional studies of Shaker channels [78–81]. Structure and mutagenesis data indicate that *κ*-PVIIA binds to Shaker via a functional dyad consisting of a pore-inserting lysine (K7) and a hydrophobic residue (F9), similar to the binding mode of other toxin blockers of potassium channels [82,83]. In an earlier computational study of the Shaker-*κ*-PVIIA complex [64], the functional dyad was reproduced but the other important interactions identified in the mutagenesis experiments [77] were not. As stressed earlier, this was due to incorrect modelling of the pore region of Shaker.

In a more recent study of *κ*-PVIIA binding to Shaker and Kv1 channels [61], a correct model of Shaker was used and the results obtained from the Shaker-*κ*-PVIIA complex model were in general agreement with the experiments. Similar protocols as in ShK were employed in creating the complex model. Snapshots of the Shaker-*κ*-PVIIA complex is shown in Figure 4. The pairs of residues involved in binding and the average distances between the pairs of atoms—obtained from the MD simulations of the complex—are given in Table 3. Comparison of the pairs of residues in Table 3 with the alanine scanning mutagenesis data [77] shows that all strongly interacting residues identified in the experiment have been accounted for in the complex structure. The binding energies of *κ*-PVIIA to Shaker and Kv1.2 have also been reproduced within chemical accuracy, providing further validation for the complex models.

**Figure 4.** Snapshots of the Shaker-*κ*-PVIIA complex showing the important residues involved in binding. The monomers A and C and B and D are shown separately for clarity.


**Table 3.** Similar to Table 2 but for the *κ*-PVIIA–Kv1 complexes. Benz refers to the COM of the benzyl group.

In contrast to Shaker, Kv1 channels are found to be insensitive to *κ*-PVIIA [75,76]. This finding can be understood by comparing the binding modes of *κ*-PVIIA to Shaker and Kv1 channels. Inspection of Table 3 shows that the hydrophobic interactions are missing in the Kv1-*κ*-PVIIA complexes. A detailed picture of the binding mode in Shaker (Figure 5) helps to explain why. The T449–D447 cross-linking keeps the threonine side chain away from the strongly coupled F9–F425 pair. Indeed the T449Y mutation breaks this cross-linking, which disrupts F9–F425 coupling and renders Shaker insensitive to *κ*-PVIIA. In a similar fashion, lack of cross-linking in the corresponding residues in Kv1.1 and Kv1.2 prevents the formation of hydrophobic interactions, making them insensitive to *κ*-PVIIA.

**Figure 5.** An alternative view of the Shaker-*κ*-PVIIA complex that demonstrates the importance of the T449–D447 cross-linking in preserving the strong F9–F425 hydrophobic interaction.

#### **4. Sodium Channel Toxins**

Computational investigation of sodium channels has just started after the first crystal structure for the bacterial channel NavAb was solved [14]. Two more bacterial Nav channels have been solved since then [84,85]. There have been some earlier model studies of Nav-toxin complexes based on potassium channels and experimental data [86–88], but their accuracy is limited. So far, binding of *μ* conotoxin to NavAb [56] and scorpion *β*-toxins to the voltage sensor [58] have been investigated. Due to substantial differences between the bacterial and mammalian Nav channels, homology modelling of the pore domain requires careful validation [89]. Here we briefly discuss binding of *μ*-conotoxin GIIIA to Nav1.4 to illustrate the differences between the toxin binding modes in potassium and sodium channels (see Figure 6 for the NMR structure of *μ*-GIIIA). *μ*-GIIIA is the first conotoxin found to block Nav channels [90], and numerous functional studies of its binding to Nav1.4 have been performed [91–97]. Thus there is a wealth of mutation data to validate the Nav1.4-*μ*-GIIIA complex models.

A homology model of the pore domain of Nav1.4 was created by aligning the DEKA residues with the corresponding EEEE residues in NavAb. The Nav1.4-*μ*-GIIIA complex was created using the same procedures as in potassium channel studies. Snapshots of the complex model (Figure 7) shows that *μ*-GIIIA interacts mainly with the outer ring EEDD residues but has no coupling to the inner ring DEKA residues. R13 makes multiple connections with residues in three domains of Nav1.4 (E403, E758, and D1532), and it is clearly the pore blocking residue, consistent with the mutation experiments [91,92]. K16 is also involved in two interactions (E758 and D1241), again in agreement with the mutation data [87,95,97]. The third important coupling is provided by the K11–D1532 interaction. The proposed model of the Nav1.4-*μ*-GIIIA complex gives a satisfactory account of the available mutation data. The binding free energy of *μ*-GIIIA is determined from the PMF calculations and has been found to agree with the experimental value within chemical accuracy. Thus the Nav1.4-*μ*-GIIIA complex has been well validated and could be used as a template in constructing homology models for the pore domain of other Nav1 channels, which are highly homologous.

**Figure 6.** NMR structure of *μ*-GIIIA [98] showing the important residues involved in binding to Nav1.4.

**Figure 7.** Snapshots of the Nav1.4-*μ*-GIIIA complex showing the important residues involved in binding. The domains D1–DIII and DII–DIV are shown separately for clarity.

It is of interest to compare the pore domains of the potassium and sodium channels, and point out the differences in toxin binding modes arising from structural constraints. As shown in Figure 8, the selectivity filter in potassium channels is very narrow, long, and has a highly negative potential. This potential attracts a Lys residue (but not the larger Arg) into the filter, which completely blocks

the channel. In sodium channels, the selectivity filter is wider and shorter, but the DEKA locus has a smaller negative potential. As a result, toxins preferentially interact with the EEDD residues in the outer ring. Because the outer ring is even wider, only an Arg residue interacting with several domains can completely block the channel—a Lys residue provides only a partial block. The wider opening in sodium channels prevents formation of a tight binding mode observed in many potassium channel-toxin complexes leading to pM affinities. Thus a crucial issue in designing drug leads for sodium channels from toxins is how to increase their relatively low affinities.

**Figure 8.** Comparison of the pore domains of Kv1.2 (yellow) and NavAb (blue) channels involved in toxin binding.
