Fig. 5: Quantitative evaluation of intracellular conformations in the WT and K227^5.40A, Y312^7.34A, C286^6.47A, and H291^6.52A mutants using MD simulations.

The populations of both the alternative conformations and those in which R156^3.50 faces toward the intracellular side decreased significantly in all mutants compared to the WT. Heatmap of the intracellular conformations based on the interatomic distance between Cα of S153^3.47 and Cζ of Y330^7.53 and the dihedral angle of C–Cα–Cγ–Cζ of Y330^7.53 for the WT and the four mutants are shown in (a–e): a WT, b K227^5.40A, c Y312^7.34A, d C286^6.47A, and e H291^6.52A. Four major states were identified (states 1–4) and further subdivided based on the orientation of R156^3.50: states 2 and 3 were split into two substates (2-A, 2-B, 3-A, and 3-B), resulting in six defined conformational states. The percentages represent the populations of each state across the three independent one-microsecond MD simulations. The percentages labeled R^3.50 indicate the proportion of structures within each state in which R156^3.50 faces toward the intracellular side. The proportions of all six conformational states for each system are summarized in (f). g–l depict representative conformations of each state observed in the WT: g State 1 (S1), canonical active conformation; h S2-A, the conformation with R156^3.50 facing intracellularly; i S2-B, similar to the canonical active conformation; j S3-A, alternative conformation with both R156^3.50 and Y330^7.53 facing intracellularly; k S3-B, other conformations in state 3; l S4, a unique conformation characterized by Y330^7.53 facing toward the intracellular side of TM1.