Figure 4.

The Thr284-Asp437 interaction is affected by both thermostabilizing mutations and the presence of Na2. Comparing the structures and simulations with paroxetine in the ABC pose, the side chain −OH of Thr284 in TM5m faces the Na2 site in the ts3 and Thr439-ts2 structures (A, C), and forms an H-bond with Asp437 in TM8m in the ts3 simulations even in the presence of the bound Na2 (B). This −OH rotates away in the Thr439-ts2 (D) and WT (F) simulations in the presence of Na2, but not in the no-Na2 condition of Thr439-ts2 (E). Note that the T439S mutation (not shown) is two residues away from Asp437, while the impact of I291A may propagate from the conserved proline-kink in the TM5 (Pro288) to Thr284. (G) The Thr284 χ1 rotamer frequency distributions in the simulations of the ts3 (black curve), Thr439-ts2 (green solid and dashed curves, for the Na2-bound and -unbound conditions, respectively), and WT (cyan curves) conditions. Its values for the ts3 and Thr439-ts2 structures are indicated by the black and green dotted lines, perpendicular to the x-axis, respectively. (H) The distances between the side chain oxygens of Thr284 and Asp437 are plotted against the distances between Na1 and Na2 for the ts3 (black), the Na2-bound Thr439-ts2 (green), and WT (cyan) conditions.