## 2 Results and Discussion
### Effect of Mutations on Free Energy Profiles of HRAS
Figure 5: FELs and representative structures of GTP-bound WT mutated HRAS: **(A)** GTP-bound WTHRAS, **(B)** GTP-bound A59 EHRAS, **(C)** GTP-bound K117R HRAS, **(D)** GDP-bound WTHRAS, **(E)** GDP-bound A59 EHRAS, **(F)** GDP-bound K117R HRAS; the left panels are FELs, the middle panels are the superimposition of two representative structures except for in the GTP-bound A59E complex, the right panels depict relative geometric positions of key residues situated at the SW1 and SW2 from the GTP- and GDP-bound WT, A59E, and K117R HRAS. The PMF is scaled in kcal/mol and the distances are indicated in Å and highlighted in red color. Regarding the GDP-associated WT HRAS, GaMD simulations identified two energy basins, EB1 and EB2 (Figure 5D, left panel). The conformations found in EB1 and EB2 accounted for 63.4% and 34.6% of the total saved conformations from the GaMD trajectories, respectively (Figure 5D, left panel). The structural changes were examined by superimposing two representative structures from EB1 and EB2. The results suggest that the switch domains SW1 and SW2 exhibited significant deviations between the two energetic states EB1 and EB2 of the GDP-bound WT HRAS. In contrast, the other domains maintained good alignment (Figure 5D, middle panel). In both energy states EB1 and EB2, the distances between D33 and E62 in the GDP-bound WT HRAS were 15.9 A (Figure 5D, right panel). Although the relative position between D33 and E62 remained unchanged, the helix \(\alpha\)2 of the SW2 was closer to the helix \(\alpha\)3 in the EB2 compared to the EB1 (Figure 5D, right panel). In the case of the GDP-bound A59E HRAS, two energy basins, EB1 and EB2, were observed (Figure 5D, left panel). The depth of these energy basins indicated a higher transition probability from the EB2 to the EB1 than that from the EB1 to the EB2. The conformations located in the EB1 and EB2 accounted for 74.2% and 24.2% of the total conformations, respectively, suggesting that most of the GDP-bound A59E HRAS conformed in EB1 (Figure 5D, right panel). To examine the structural differences, two representative structures from EB1 and EB2 were aligned. The results revealed distinct deviations in the SW1 between the two energy states, while the SW2 in the GDP-bound A59E HRAS appeared to be more ordered than the GDP-bound WT HRAS (Figure 5E, left panel). The distances between D33 and E62 in the EB1 and EB2 states of the GDP-bound A59E HRAS were 16.5 A and 26.8 A, respectively (Figure 5E, right panel). This finding indicates that the A59E mutation results in a looser switch domain compared to the GDP-bound WT HRAS, providing evidence for the impact of A59E on the binding of HRAS to its effector. As for the GDP-bound K117R HRAS, GaMD simulations also revealed the presence of two energy basins, EB1 and EB2 (Figure 5F, left panel). The depth of the EB1 was found to be greater than that of the EB2 in the GDP-bound K117R HRAS. It was observed that 63.4% of the total conformations saved in the GaMD trajectories were trapped in the EB1. In comparison, 34.6% were observed in the EB2, indicating that the majority of conformations of the GTP-bound K117R HRAS were predominantly distributed in the EB1. A comparison of the EB1 and EB2 superimposed structures, representative of the GDP-associated HRAS (Figure 5F, middle panel), revealed significant deviations in the SW1 and the loop L3, which may potentially impact HRAS-effector binding and allosteric regulation. The distances between D33 and E62 in the GDP-bound K117R HRAS located in the EB1 and EB2 were measured to be 18.3 A and 18.2 A, respectively (Figure 5F, right panel), a considerable increase compared to the corresponding distances in the GDP-bound WT HRAS. These findings suggest that the K117R mutation induces a looser switch domain than the WT HRAS, which likely affects the binding between HRAS and its effector. Based on the analyses conducted, it is evident that the two mutations, A59E and K119R, significantly impact the conformational states of HRAS, particularly in the switch domains.