Figure 5.

Impact of Na⁺ binding and Glu-290 protonation on the extent of the outward-open character of LeuT conformations. (A) The extents of outward-open character of various configurations are reflected by the normalized distributions of the number of water molecules in the EV. Compared with the occluded state with S1-bound substrate (black, based on a 230 ns MD trajectory described previously (3,32)), the Na⁺-only configuration (green, 60–420 and 720–960 ns periods of MD1 for the left and right panels, respectively) has ∼30 more waters in the EV, and the absence of Na1 (red, 480 ns) further opens the EV slightly. The removal of the charge of Glu-290 (purple, 360 ns) renders the EV less outward-open. Consistent trends are observed from the no-Na1 simulations started from both 60 (left) and 720 (right) ns points of MD1 (see text). The multiple peaks of the green curve in the left panel indicate that the MD1 is in a conformational transition in the 60–420 ns period. The distribution plots were generated from the water count data using a Gaussian kernel density estimate (density function in R (18)). (B) The roles of Na⁺ binding and Glu-290 protonation in the control of conformational dynamics of the transport cycle. The configurations are in the same color code as in panel A.