3.5 | Model of the bupivacaine and ropivacaine binding mode in the central cavity and the ‘side pockets´ of Kv1.5
To determine the binding mode of bupivacaine and ropivacaine in Kv1.5 channels, we used in silico docking experiments and molecular dynamics (MD) simulations of charged S‑bupivacaine and ropivacaine, respectively, using a Kv1.5 homology model based on the rKv1.2-Kv2.1 chimera crystal structure (Figure 4). After docking of Kv1.5 in complex with five ligands, one in the central cavity and one in each ‘side pocket´, we performed 100 ns molecular dynamics (MD) simulations and selected 200 complexes for each system (a pose every 0.1 ns from the last 20 ns) to perform a clustering analysis. According to this analysis we identified highly populated clusters in the central cavity and the ‘side pockets´, for both local anesthetics (Figure 4 and Supplementary Table 1). This indicates that in general, both local anesthetics adopt a fairly stable conformation during the simulation in the central cavity and in the ‘side pockets´, since the population of the clusters provides a measure for the stability of the ‘drug to receptor´ interaction. The most populated clusters contained in all cases more than 50% of the total number of 200 conformers. For both local anesthetics, some clusters were even formed by all or almost all ligand conformers (central cavity and in some of the ‘side pockets´, see Supplementary Table 1).
The proposed binding mode of bupivacaine in the central cavity is illustrated in Figure 4b, illustrating a cluster containing 198 out of 200 conformers. The ropivacaine binding mode is illustrated in Figure 4c, containing 199 out of the 200 conformers. Both local anesthetics interact with Kv1.5 in a similar mode (Figure 4b,c). For both compounds the most populated cluster (Figure 4b,c) provided the basis for interactions with the residues identified in the alanine mutagenesis scan of the S6 segment (Figure 1g, 3g), except for L510 as this residue is facing into the ‘side pockets´.
Figure 4d,f illustrates the two most likely binding modes of bupivacaine in the ‘side pockets´ of Kv1.5, representing the two most occupied clusters (containing 200 and 199 conformers). The two most occupied ‘side pockets´ clusters (containing 196 and 182 conformers) derived from the MD simulations with ropivacaine are illustrated underneath (Figure 4e,g). Note that the two most occupied clusters of bupivacaine are located at the opposite end of the ‘side pockets´ (Figure 4d,f) compared to ropivacaine (Figure 4e,g). Consistent with our alanine-scanning mutagenesis, ropivacaine interacts with the S4 segment, residues of the proximal S4-S5 linker and the backside of the S5 and S6 segment of a neighbouring subunit (Figure 4e,g). In contrast, bupivacaine does not bind next to the S4 segment and is interacting with residues of the S5 segment of the same subunit (Figure 4d,f), supporting our initial alanine mutagenesis screen of the S4 and S4-S5 linker in which we did not observe any effects on bupivacaine inhibition (Marzian, Stansfeldet al. , 2013) (Figure 1g). Thus, both local anesthetics bind to the central cavity and the ‘side pockets´, albeit binding to the ‘side pockets´ can occur in two different regions (Figure 5) which might be involved in tuning stereoselectivity and affinity of Kv1 channel inhibition by local anesthetics.