## 2 Results and Discussion
### Analyses of GTP- and GDP-HRAS Interaction Networks
The non-bonded interactions of GTP (**A**) and GDP (**B**) with HRAS; the electrostatic interactions of magnesium ion Mg\({}^{2+}\) with residues and GTP (**C**) and GDP (**D**), respectively. and K147 in loop L4 of WT HRAS (Figure 6A,B). These HBIs, except for those involving K117, had occupancies larger than 52.8% and remained stable throughout the entire GaMD simulations of GTP- and GDP-bound WT HRAS (Table 1). The occupancies of four HBIs between GTP and residues V29, D30, Y32, and T35 from the switch region SW1 in WT HRAS exceeded 60.0%. In comparison, the occupancies of three HBIs between GDP and residues V29, D30, and Y32 ranged from 29.8% to 39.7% (Table 1), suggesting weaker HBIs between SW1 and GDP compared to GTP. When comparing to GTP- and GDP-bound WT HRAS, the A59E mutation resulted in a noticeable reduction in the occupancy of HBIs between GTP and residues V29, D30, Y32, T35, and D119, as well as a decrease in HBIs between GDP and residues G13, G15, A18, V29, D30, Y32, D119, S145, and K117. However, A59E significantly increased the occupancy of HBIs between GDP and V14 and S17 (Table 1). These findings indicate that A59E had a more significant impact on HBIs between GDP and HRAS than between GTP and HRAS. On the other hand, the K117R mutation substantially decreased the occupancy of HBIs between GTP and residues G13, S17, V29, D30, Y32, T35, N116, D119, and K147. It also reduced the occupancy of HBIs between GDP and residues G13, K16, A18, V29, D30, Y32, D119, and A146. Furthermore, K117R abolished the HBIs of GTP and GDP with residue 117. Changes in the stability of HBIs between GTP, GDP, and HRAS caused by A59E and K117R can impact the activity of HRAS. Based on Figure 6A, GTP formed two salt bridge interactions with K16, a \(\pi\)-\(\pi\) interaction with F28, and a salt bridge interaction with D119. On the other hand, GDP only formed a salt bridge interaction with K16 while maintaining the same interactions with F28 and D119 as those observed in GTP (Figure 6B). The distances involved in the salt bridge interactions of GTP and GDP were calculated using the GaMD trajectories. The time courses and frequency distributions of these distances are illustrated in Figures 7 and S7. The temporal evolution of the distances between GTP and K16 for the two salt bridge interactions demonstrates that these salt bridges exhibited remarkable stability throughout the GaMD simulations. Specifically, in the GTP-bound WT, A59E, and K117R HRAS, the distances between the nitrogen atom NZ of K16 and the phosphorus atom PB of GTP were distributed at 3.35, 3.41, and 3.35 A, respectively (Figure 7A). Furthermore, the consistent population of distances between the nitrogen atom NZ of K16 and the phosphorus atom PG at 3.71 A (Figure 7A) suggests that the A59E and K117R variants had minimal impact on the stability of the two salt bridge interactions with K16. While the distances between the mass centers of the phenyl ring in F28 and the guanine group of GTP remained within a stable fluctuation range (Figure S7A), their frequency distributions revealed that the GTP-bound A59E HRAS exhibited two peak values (Figure 7A). This indicates that K117R had little influence on the \(\pi\)-\(\pi\) interaction between GTP and F28, while A59E slightly weakened this interaction compared to the GTP-bound WT HRAS. The time evolution of the salt bridge interaction distance between GTP and D119 suggests that this distance remained highly stable (Figure S7A), with both the GTP-bound WT and mutated HRAS exhibiting distributions centered around 3.45 A. Consequently, A59E and K117R had minimal impact on the stability of this salt bridge. In contrast, the distance between the nitrogen atom NZ of K16 and the phosphorus atom PA showed significant fluctuation over the simulation time (Figure S7B), with most of its frequency distribution located at 6.19 A (Figure 7B). This indicates that the phosphorus atom PA of GDP did not form a salt bridge interaction with the nitrogen atom NZ of K16; instead, it only yielded electrostatic interactions.