Figure 1.
The thermodynamic cycle used in the free energy calculations.
The superscript 0 denotes amino acids with no charges on the side chain atoms.
Figure 2.
The K18A mutation does not change the binding modes of ShK to Kv1.1 and Kv1.3 channels.
Snapshots of ShK[K18A] (yellow with colored side chains) and ShK (transparent orange) in complex with Kv1.1 and Kv1.3 are superposed to show that there is a good overlap between the toxin backbones, and all the important interactions identified in the ShK complexes are preserved in the ShK[K18A] complexes. To give the full picture, two views of the cross sections of the complex, depicting the monomers C and A (left panel) and D and B (right panel) are presented. Only the residues involved in binding are indicated explicitly.
Table 1.
Comparison of the strongly interacting pair distances in the ShK–Kv1.x complexes with those in the ShK[K18A] –Kv1.x complexes.
Figure 3.
Convergence of the FEP and TI calculations.
(A) Convergence of the FEP calculations for discharging/charging of the K18 in ShK in the binding site/bulk. Running averages of ΔGi for windows at λ = 0 (black), λ = 0.2 (red), λ = 0.4 (green), λ = 0.6 (blue), λ = 0.8 (yellow) and λ = 0.999 (magenta) are plotted as a function of the production time for Kv1.1 (left) and Kv1.3 (right). (B) Similar to A but showing the convergence of the FEP calculations for transformation of the uncharged Lys side chain to that of Ala. (C) Convergence of the TI calculations for discharging/ charging of K18 in ShK in the binding site/bulk. Running averages of the ΔG values obtained from the TI calculations are plotted as a function of the production time for Kv1.1 (left) and Kv1.3 (right). Both the forward (black) and the negative of the backward (red) results are shown to check for hysteresis effects, which remain well below 1 kcal/mol for both channels.
Table 2.
Differences in the binding free energy of ShK to Kv1.1 and Kv1.3 due to the K18A mutation, calculated with the FEP and TI methods.
Figure 4.
Comparison of the PMFs for the unbinding of ShK and ShK[K18A] from the Kv1.1 and Kv1.3 channels.
Table 3.
The relative and absolute binding free energies obtained from the PMF calculations for the Kv1.x–ShK and Kv1.x–ShK[K18A] complexes.
Table 4.
Comparison of the binding free energy differences for Kv1.1 and Kv1.3, and the selectivity free energy for Kv1.3/Kv1.1, obtained using the FEP, TI and PMF methods, with the experimental results.
Figure 5.
ShK[K18A] is selective for Kv1.3 over Kv1.1 channels and preferentially targets TEM lymphocytes in vitro and in vivo.
(A) Effects of ShK (▪) and ShK[K18A] (○) on Kv1.3 currents measured by whole-cell patch-clamp on L929 fibroblasts stably transfected with mKv1.3. The two left panels show whole-cell Kv1.3 currents before (control) and after perfusion of ShK[K18A] (left panel) or ShK (middle panel). The panel on the right shows the Kv1.3 currents remaining after steady-state block is reached with the different concentrations of ShK and its analog, fitted to a Hill equation (N = 5–6 cells per concentration).(B) Effects of ShK (▪) and ShK[K18A] (○) on Kv1.1 currents measured by whole-cell patch-clamp on L929 fibroblasts stably transfected with mKv1.1. The two left panels show whole-cell Kv1.1 currents before (control) and after perfusion of ShK[K18A] (left panel) or ShK (middle panel). The panel on the right shows the Kv1.1 currents remaining after steady-state block is reached with the different concentrations of ShK and its analog, fitted to a Hill equation (N = 6–7 cells per concentration).(C) Effects of ShK (▪) and ShK[K18A] (○ and dashed line) on the proliferation of rat TEM cells measured in vitro by the incorporation of [3H] thymidine into the DNA of dividing cells (N = 3). (D) Effects of ShK (▪) and ShK[K18A] (○ and dashed line) on the proliferation of rat splenocytes (mainly naïve/TCMcells; N = 3).(E) Effects of the subcutaneous administration of 100 µg/kg ShK[K18A] on an active DTH reaction elicited against ovalbumin. Data show the difference in challenged and non-challenged ear thickness in vehicle-treated rats and ShK[K18A]-treated rats (N = 6/group).