In nitrogen of Lys23 and the carbonyl group of Gly376 is above the upper boundary of the distance restraint. Otherwise, the force is zero if the distance between Lys23-Gly376 is lower than the upper boundary. In the presence of the distance restraint, the toxin is gradually drawn to the outer vestibule of Kv1.2. Figure 2A displays a representative configuration showing the position of MTx relative to the outer vestibule of Kv1.2 after the docking simulation totaling 20 ns. Two key contacts of the MTx-Kv1.2 complex are shown. One firm contact is inside the selectivity filter, where Lys23 of MTx forms hydrogen bonds with the carbonyl groups of Tyr377 from the four channel subunits. A hydrogen bond is considered to be formed if the donor and acceptor atoms are within 3 A˚ of each other and the donor-hydrogen-acceptor angle is $150u. The other contact is between Arg14 of MTx and Asp355 on the P-loop turret of Kv1.2, where these two residues form a hydrogen bond and salt bridge. A salt bridge is considered to be formed if the distance is less than 4 A˚ between a side chain oxygen atom from an acidic residue and a nitrogen atom from a basic residue. Figure 2B shows that Lys7 of MTx forms the third strong contact with Asp363 on the outer vestibular wall of Kv1.2. The Lys7- Asp363 appears to be less stable than Arg14-Asp355; Lys7 occasionally forms a hydrogen bond with Gln357 in the P-loop turret. Figure 3 shows the lengths of the salt bridges Arg14-Asp355 and Lys7-Asp363 as a function of the simulation time over the last 15 ns. The Lys7-Asp363 salt bridge forms at 10 ns but breaks at 15 ns, whereas the Arg14-Asp355 salt bridge remains stable between 10 and 20 ns. In the second and third docking simulations, the two salt bridges were also observed to form and break. Thus, the simulations show that the interactions between the MTx and the outer vestibule are highly dynamic, although Lys23 persistently occludes the ion conduction pathway. Similar dynamic toxin-channel interactions have been observed in previous simulations of ChTx and Kv1.3. Double mutant cycle analysis has also suggested that Arg14 may be coupled with Asp355. Our model displayed in Figure 2 is consistent with mutagenesis experiments, which suggest that Arg14 is coupled with Asp355, and Lys7 is coupled with Asp363. We note that two acidic residues Asp352 and Glu353 are in close proximity to Asp355. These two residues could form salt bridges with MTx if Asp355 is mutated to a neutral or basic amino acid. This would explain the minimal effect on MTx binding affinity caused by the alanine mutation of Asp355 observed experimentally. Thus, our model of MTx-Kv1.2 is in accord with the experimental measurements of Visan et al. To further VE-821 verify the binding mode of MTx-Kv1.2 predicted by the docking using biased MD simulation, molecular docking calculations using the rigid-body docking program ZDOCK are performed. This docking program has been applied to numerous similar toxin-channel systems. We assume that the docking pose generated by the program is correct if Lys23 protrudes into the selectivity filter. If the same MTx structure as that used in biased MD were used in ZDOCK, the correct docking pose obtained is found to be nearly identical to that predicted from biased MD. The survival, development, phenotype, and function of these immune cells are modulated by contact-dependent and independent signals from the microenvironment.