## 2 Results and Discussion
### Effect of Mutations on Free Energy Profiles of HRAS  
It is well known that free energy profiles can provide significant information to deeply understand the influences of A59E and K117R on conformational changes in the GTP- and GDP-bound HRAS. We constructed free energy landscapes (FELs) using two reaction coordinates: the distance between the C\(\alpha\) atoms of residue D33 in the SW1 and residue E62 in the SW2, as well as the RMSDs of heavy atoms from HRAS (Figure 5). The distance can effectively capture conformational transitions between SW1 and SW2, while the RMSDs accurately represent the overall structural fluctuations of HRAS. The reason we chose them as reaction coordinates is precisely because of this. In the GTP-bound WT HRAS, GaMD simulations captured two energy basins, EB1 and EB2 (Figure 5A, left panel). The EB1 basin was deeper than EB2, indicating that the conformational transition from EB2 to EB1 in the GTP-bound WT HRAS is more accessible than the reverse transition. The conformations at EB1 and EB2 accounted for 81.2% and 17.8% of the total conformations, respectively (Figure 5A, left panel). Therefore, the GTP-bound WT HRAS conformations were mostly found in the EB1. Two representative structures falling into EB1 and EB2 were aligned (Figure 5A, left panel) to reveal structural differences. The SW1 from the GTP-WT HRAS complex exhibited significant deviations between the EB1 and EB2, whereas the remaining structures of the GTP-bound WT HRAS were aligned very well (Figure 5A, right panel). The distances from D33 to E62 were 15.6 and 26.0 A in the EB1 and EB2 states, respectively, in the GTP-associated WT HRAS. These findings suggest that the GTP-bound WT HRAS switch domains are located at a tighter state in the EB1 than in the EB2. For the GTP-bound A59E HRAS, only one energy basin, EB1, was detected by GaMD simulations (Figure 5B, left panel), and the representative structure of the GTP-bound A59E located in the EB1 was extracted from the GaMD trajectory (Figure 5B, middle panel). Compared to the GTP-bound WT HRAS, A59E led to a less energetic state, implying that A59E induces conformational rearrangement of HRAS. At this state, the distance between D33 and E62 was 18.2 A (Figure 5B, right panel), which is different from the switch state of the GTP-bound WT HRAS. For the GTP-bound K117R HRAS, GaMD simulations revealed the presence of two primary energy basins, EB1 and EB2 (Figure 5C, left panel), with EB1 being significantly more profound than EB2. Consequently, the transition probability from the EB2 to the EB1 was much higher than the reverse transition from the EB1 to the EB2. The conformations located at EB1 and EB2 accounted for 88.3% and 10.3% of the total structures, respectively, indicating that most of the conformations of the GTP-bound K117R HRAS are distributed in the EB1 basin. The two representative structures located at EB1 and EB2 were superimposed, as shown in Figure 5C (middle and left panels). The results indicate that the K117R mutation caused a noticeable deviation of the loop L2 compared to the GTP-bound WT HRAS. Interestingly, the loop L2 was located near the allosteric position of HRAS, suggesting that K117R may affect the allosteric regulation of HRAS activity. In the EB1 state of the GTP-bound K117R HRAS, the distance between D33 and E62 was 16.2 A, while in the EB2 state, it was 28.9 A (Figure 5C, middle and left panels). Therefore, the switch domains of the GTP-bound K117R HRAS are looser than those of the GTP-bound WT HRAS, indicating that the K117R mutation influences the binding of HRAS to its effectors.