Predefined residue pairs below one or several predefined upper boundaries. For MTx, both theoretical [16,17] and experimental [5,7] studies are consistent with Lys23 occluding the selectivity filter of Kv1.2. In addition, Lys23 is located in the b-strand immediately preceded by the a-helix (Figure 1A), corresponding to the position of the key lysine residue found in closely related toxins [27,28]. Thus, an interaction residue pair between Lys23 of MTx and Tyr377 in the selectivity filter of Kv1.2 is identified. The available knowledge on the Lys23-Tyr377 residue pair enables us to use biased MD to predict the position of MTx relative to the outer vestibule of Kv1.1, Kv1.2 and Kv1.3 on block.Figure 1. Structure of MTx and Kv1.2. (A) The secondary structure of MTx (PDB ID 1TXM [32]). a-Helix is shown in purple and b-sheets in yellow. The side chains 
of four key buy CASIN residues are highlighted. (B) Sequence alignment of the pore domains of Kv1.1, Kv1.2 and Kv1.3. Key different residues are highlighted in green. The P-loop turret and selectivity filter regions are indicated with 15481974 horizontal bars. Numbering is that of Kv1.2. (C) Pore domain of Kv1.2 (PDB ID 3LUT [30]) viewed perpendicular to the channel axis. Two channel subunits are shown. The side chain of Asp355 and the backbone carbonyl groups of Thr373 and Asp379 are highlighted. Green spheres represent the two K+ ions in the selectivity filter. The two water molecules in the selectivity filter are also shown. doi:10.1371/journal.pone.0047253.MedChemExpress JI 101 gMolecular Dynamics SimulationsWe construct homology models of the pore domains of human Kv1.1 and Kv1.3 channels on the crystal structure of rat Kv1.2 [29,30] using the methods detailed elsewhere [20,31]. These three channels share .90 sequence identity in the pore domain. Human and rat Kv1.2 channels differ only at position 355 in the pore domain, where it is a glutamate in rat and an aspartate in human. Each channel is then embedded in a 2oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine bilayer (,80 lipids/leaflet) and a box of explicit water (,15,000 molecules). Approximately 58 K+ and Cl- ions each are added into each system, corresponding to a salt concentration of 0.2 M. Two K+ ions are moved to the S2 and S4 binding sites of the selectivity filter, with the S1 and S3 ion binding sites occupied by two water molecules (Figure 1C). Each system is equilibrated for ?5 ns, and the sizes of the simulation boxes are ,856856105 A3 after the 5-ns simulation. In our previous work [31], we have shown that the P-loop turrets of Kv1.1 and Kv1.2 move outward substantially in the presence of the voltage-sensing domain. Thus, to model the P-loop turret correctly in the reduced channel model in which the voltage-sensing domain is truncated, the centers of mass of the residues at position 355 in the P-loop turrets of Kv1.1 and Kv1.2 (Figure 1B) are ?maintained to be at least 19?0 A from the channel central axis. After the 5-ns equilibration, we 12926553 place MTx (PDB ID 1TXM ?[32]) into each simulation box, ,15 A above the position where the toxin is fully bound. A flat-bottom harmonic distance restraint is applied between the side chain nitrogen atom of MTx-Lys23 and the carbonyl group of the channel residue Gly376. When Lys23 protrudes into the selectivity filter and forms hydrogen ?bonds with Tyr377, the side-chain nitrogen is ,4 A above theExperimental studies performed on different types of cells expressing Kv1.1-Kv1.3 channels have demonstrated that MTx is selective for.Predefined residue pairs below one or several predefined upper boundaries. For MTx, both theoretical [16,17] and experimental [5,7] studies are consistent with Lys23 occluding the selectivity filter of Kv1.2. In addition, Lys23 is located in the b-strand immediately preceded by the a-helix (Figure 1A), corresponding to the position of the key lysine residue found in closely related toxins [27,28]. Thus, an interaction residue pair between Lys23 of MTx and Tyr377 in the selectivity filter of Kv1.2 is identified. The available knowledge on the Lys23-Tyr377 residue pair enables us to use biased MD to predict the position of MTx relative to the outer vestibule of Kv1.1, Kv1.2 and Kv1.3 on block.Figure 1. Structure of MTx and Kv1.2. (A) The secondary structure of MTx (PDB ID 1TXM [32]). a-Helix is shown in purple and b-sheets in yellow. The side chains of four key residues are highlighted. (B) Sequence alignment of the pore domains of Kv1.1, Kv1.2 and Kv1.3. Key different residues are highlighted in green. The P-loop turret and selectivity filter regions are indicated with 15481974 horizontal bars. Numbering is that of Kv1.2. (C) Pore domain of Kv1.2 (PDB ID 3LUT [30]) viewed perpendicular to the channel axis. Two channel subunits are shown. The side chain of Asp355 and the backbone carbonyl groups of Thr373 and Asp379 are highlighted. Green spheres represent the two K+ ions in the selectivity filter. The two water molecules in the selectivity filter are also shown. doi:10.1371/journal.pone.0047253.gMolecular Dynamics SimulationsWe construct homology models of the pore domains of human Kv1.1 and Kv1.3 channels on the crystal structure of rat Kv1.2 [29,30] using the methods detailed elsewhere [20,31]. These three channels share .90 sequence identity in the pore domain. Human and rat Kv1.2 channels differ only at position 
355 in the pore domain, where it is a glutamate in rat and an aspartate in human. Each channel is then embedded in a 2oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine bilayer (,80 lipids/leaflet) and a box of explicit water (,15,000 molecules). Approximately 58 K+ and Cl- ions each are added into each system, corresponding to a salt concentration of 0.2 M. Two K+ ions are moved to the S2 and S4 binding sites of the selectivity filter, with the S1 and S3 ion binding sites occupied by two water molecules (Figure 1C). Each system is equilibrated for ?5 ns, and the sizes of the simulation boxes are ,856856105 A3 after the 5-ns simulation. In our previous work [31], we have shown that the P-loop turrets of Kv1.1 and Kv1.2 move outward substantially in the presence of the voltage-sensing domain. Thus, to model the P-loop turret correctly in the reduced channel model in which the voltage-sensing domain is truncated, the centers of mass of the residues at position 355 in the P-loop turrets of Kv1.1 and Kv1.2 (Figure 1B) are ?maintained to be at least 19?0 A from the channel central axis. After the 5-ns equilibration, we 12926553 place MTx (PDB ID 1TXM ?[32]) into each simulation box, ,15 A above the position where the toxin is fully bound. A flat-bottom harmonic distance restraint is applied between the side chain nitrogen atom of MTx-Lys23 and the carbonyl group of the channel residue Gly376. When Lys23 protrudes into the selectivity filter and forms hydrogen ?bonds with Tyr377, the side-chain nitrogen is ,4 A above theExperimental studies performed on different types of cells expressing Kv1.1-Kv1.3 channels have demonstrated that MTx is selective for.
