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. 2021 Feb 18;296:100430. doi: 10.1016/j.jbc.2021.100430

Figure 5.

Figure 5

TM4-9 mutations alter the conformational dynamics of the transporter in favor of the IF → occluded → OF transition. Snapshots at t = 0 (left) and 100 ns (middle: viewed from EC region; right: side view) observed in MD simulations of (A) outward-facing open (OFo) wt DAT, (B) OFo TM4-9, (C) inward-facing open (IFo) wt DAT, and IFo (D) TM4-9 mutant. In the simulations of substrate/ion binding by the wt DAT in the OF state (A), the hydrophobic interaction between L459 and F154 facilitates the rotational isomerization of F155 in favor of the closure of the EC gate Y156-F320, thus promoting the progress to the occluded state. The mutation I248F in TM4-9 mutant leads to π-stacking interactions between I248F and F457, which stabilize the OF state (B; right), and the EC gate F320-Y156 remains open (B; middle). In the IF state (C–D), the hydrophobic interaction between L459 and F154 which stabilizes the WT DAT IF state is weakened in the mutant (where L459 is replaced by Gly). There is an increased flexibility at this region, and F155 undergoes a rotational isomerization such that the inner gate-forming residues Y156 and F320 open; the mutation thus facilitates the opening of the EC gate or the transition from IF toward the OF state. Cumulative histograms of the center of mass (COM) distances between (E) inner EC gate residues Y156 and F320; and (F) F155 and F320 observed in the simulations of the wt DAT OF (orange with white line), TM4-9 OF (lime with white line), wt DAT IF (cyan with black line) and TM4-9 IF (purple with black line), based on three runs for each OF conformer and two for IF conformer, each of duration 200 ns. In both cases, the mutant exhibits a transition towards larger values indicated by the colored arrows. G, The shape of GNM mode 1. The residues with values close to zero refer to structural regions that serve as hinges in the global mechanics of the transporter. Note that F154-F155, I248-V249, and L458-L459 serve as hinges (shown in red squares). Calculations in G were performed using ProDy. H, The residues participating in the global hinge, identified in G, displayed in vDW format, based on DAT OF model. DAT, dopamine transporter; GNM, Gaussian network model; IF, inward-facing; OF, outward-facing; TM, transmembrane.