## 2 Results and Discussion
### Analyses of GTP- and GDP-HRAS Interaction Networks
Furthermore, K117R slightly weakened this electrostatic interaction. On the other hand, the distance between the nitrogen atom NZ of K16 and the phosphorus atom PB of GDP exhibited a stable fluctuation (Figure 4B), with values of 3.53, 3.47, and 3.63 A observed in the GDP-bound WT, A59E, and K117R HRAS, respectively (Figure 7B). This suggests that the nitrogen atom NZ of K16 formed a salt bridge with the phosphorus atom PB of GDP. Thus, K117R slightly disrupted the salt bridge interaction between GDP and K16 compared to the GDP-bound WT HRAS. According to Figure 7B, the phenyl ring of F28 engaged in a \(\pi\)-\(\pi\) interaction with the hydrophobic ring of GDP, and the distances involved in this interaction exhibited stable fluctuations (Figure S7B). As depicted in Figure 7B, the distances between the mass centers of the phenyl group in F28 and the hydrophobic ring of GDP were approximately 5.11 A in the GDP-bound WT and K117R HRAS variants. In contrast, the GDP-bound A59E HRMS exhibited two peak values and a wider distribution for this distance (Figure 7B). Consequently, K117R minimally influenced the \(\pi\)-\(\pi\) interaction between GDP and F28, while A59E weakened this interaction relative to the GDP-bound HRMS. The distances corresponding to the salt bridge interaction between GDP and D119 fluctuated within a stable range in both the GDP-bound WT and mutated HRMS (Figure S7B), with their frequency distributions largely overlapping (Figure 7B). This suggests that A59E and K117R did not significantly affect the salt bridge interaction between GDP and D119. To investigate the effects of mutations on the stability of Mg\({}^{2+}\), we analyzed the distances of Mg\({}^{2+}\)-mediated interactions using the CPPTRA] program. The results are presented in Figures 7 and S8. The distances between Mg\({}^{2+}\) and the oxygen atom OG of S17 exhibited stable fluctuations during the GaMD simulations of the GTP-bound WT, A59E, and K117R HRAS variants (Figure S8A). The frequency distributions of these distances overlapped (Figure 7C), indicating that A59E and K117R had minimal impact on the interaction between Mg\({}^{2+}\) and S17. In contrast, the distances between Mg\({}^{2+}\) and the oxygen atom OIG of T35 fluctuated significantly during the GaMD simulations of all three systems (Figure S8A). The frequency distributions suggest that A59E weakened the electrostatic interaction between Mg\({}^{2+}\) and OIG compared to the GTP-bound WT HRMS, while K117R strengthened this interaction (Figure 7C). Furthermore, the distance between Mg\({}^{2+}\) and the mass center of the oxygen atoms OD1 and OD2 of D57 was highly unstable during the GaMD simulations of the GTP-bound WT and mutated HRMS (Figures 7C and S8A). K117R significantly reduced the electrostatic interaction between Mg\({}^{2+}\) and the carbonyl group of D57. The distances between Mg\({}^{2+}\) and the phosphorus atoms PB and PG of GTP showed stable and narrow fluctuations throughout the simulations of the three systems (Figure S8A). Their frequency distributions almost overlapped (Figure 7C), indicating that A59E and K117R had minimal impacts on the electrostatic interactions between Mg\({}^{2+}\) and GTP. In Figure S8B, it can be observed that the distances between Mg\({}^{2+}\) and the oxygen atom OG of S17 were highly unstable during the simulations of the GDP-bound WT, A59E, and K117R HRMS. The GDP-bound WT HRMS frequency distribution display a single peak, while the GDP-bound A59E and K117R HRMS show two peaks. Furthermore, the second peak in the mutated HRMS is shifted to the right (Figure 7D), suggesting that A59E and K117R weakened the electrostatic interactions between Mg\({}^{2+}\) and OG of S17. According to Figure S7B, the distances of Mg\({}^{2+}\) from the oxygen atom O1G of T35 in switch region SW1, as well as the mass center of the oxygen atoms OD1 and OD2 from D57 near switch region SW2, exhibited high unordered fluctuations (Figure S8B), which is supported by their frequency distributions with multiple peaks (Figure 7D).