FIGURE 6.

Effect of S205L on stability of the open state conformation. A, molecular dynamics simulations of the single-mutant (R129S) and double-mutant (R129S/L205S) structures, i.e. where residues in the mutant structure were converted back to wild-type serine. Left, the pore radius, calculated by HOLE (40) along the pore axis, is shown for the HBC at Tyr¹³². The radius of a hydrated K⁺ ion is indicated by the dotted blue line. For times t = 0 ns and t = 100 ns, snapshots of the respective locations obtained in the R129S/L205S simulation are shown (right). B, as shown for A, but the radius was calculated at the position of the G-loop (Gln²⁵²). Although the G-loop closes only in the R129S/L205S simulation, the side chains of the HBC close the pore in both simulations. C, overlay of the CTD of the S129R/S205L mutant structure with a snapshot at t = 100 ns of the R129S/L205S simulation. No significant rotation of the CTD is observed. D, comparison of the intersubunit interface in the R129S/L205S simulation. At 0 ns, the side chains of Glu¹⁷³ and Asp¹⁷⁵ point toward the pore, and His¹⁷⁷ points down, interacting with Asp¹⁹⁷ and Arg²⁰². At 100 ns, His¹⁷⁷ has adopted the closed state conformation, and the side chains of Glu¹⁷³ and Asp¹⁷⁵ turn into the outward-facing position, also adopting the closed state orientation. E, APBS profiles of S129R and S129R/S205L. The Poisson-Boltzmann potential energy of a K⁺ ion, calculated along the pore axis of the CTD, is markedly lower in S129R/S205L due to reorientation of Glu¹⁷³ and Asp¹⁷⁵, rendering passage of K⁺ through the CTD pore more energetically favorable. F, number of cations (Na⁺) in the cytoplasmic pore (−16 Å ≤ z ≤ −37 Å) during the R129S/L205S simulation. G, number of cations (Na⁺) in the cytoplasmic pore during the R129S simulation.