## 2 Results and Discussion
### Effect of A59E and K117R on Dynamics of HRAS
In the GDP-bound inactive HRAS, K117R remarkably enhances the structural fluctuation of HRAS along the first eigenvector compared to the GDP-bound WT HRAS, but A59E reduces the structural fluctuation of HRAS along the first eigenvector (Figure S6B). The above analyses imply that these two mutations possibly affect the activity of HRAS. To further explore the effects of A59E and K117R on the dynamics behavior of HRAS, the first eigenvector resulting from the PCA is visualized in Figure 4. GTP. The GDP and mutation sites are also depicted in Figure 4. The results indicate that two switch domains SW1 and SW2 in HRAS show high structural flexibility and that mutations heavily affect  
Figure 3: DCCMs calculated using the coordinates of the C\(\alpha\) atoms in HRAS: (**A**) corresponding to the DCCMs of the GTP-bound WT HRAS, and (**B**) corresponding to the DCCMs of the GDP-bound WT HRAS. The differences in DCCMs between the GTP/GDP-bound mutants and the GTP/GDP-bound WT HRAS: (**C**) between GTP-bound A59E and GTP-bound WT HRAS, (**D**) between GDP-bound A59E and GDP-bound WT HRAS, (**E**) between GTP-bound K17R and GTP-bound WT HRAS, and (**F**) between GDP-bound K117R and GDP-bound WT HRAS. their motion behavior. Compared to the GTP-bound WT HRAS (Figure 4A), A59E not only leads to more disordered states of the SW1, as indicated by unordered arrow directions, but also inhibits the fluctuation amplitude of the SW2 along the first eigenvector (Figure 4C). Meanwhile, A59E changes the motion direction of the L3 loop (Figure 4C). By referencing the GTP-bound WT HRAS (Figure 4A), K117R not only results in the SW2 moving in the opposite direction, but also strengthens the fluctuation amplitude of the SW1 along the first eigenvector (Figure 4E). In addition, K117R also alters the movement direction of the L3 loop relative to the GTP-bound WT HRAS (Figure 4E). By comparison with the GDP-bound WT HRAS (Figure 4B), A59E completely changes the relative motion direction of the helix \(\alpha\)3 to the SW2. Meanwhile, it obviously weakens the fluctuation of the SW2 and \(\alpha\)3, which possibly generates an effect on the allosteric regulation (Figure 4D). Furthermore, A59E also changes the motion direction of the L3 loop and the SW1 compared to the GDP-bound WT HRAS (Figure 4D). In the GDP-bound K117R HRAS, the motion direction of the helix \(\alpha\)3 and the loop L3 are hardly changed due to K117R relative to the GDP-bound WT HRAS, and their fluctuation amplitudes are slightly weakened because of K117R (Figure 4F). More interestingly, the motion directions of the SW1 and SW2 are altered by K117R; furthermore, the fluctuation amplitudes of these switch domains are also decreased compared to the GDP-bound WT HRAS. Thus, A59E and K117R significantly influence the dynamics behavior of the SW1, SW2, L3, and \(\alpha\)3 in the GTP- and GDP-bound HRAS. Based on the analyses described above, A59E and K117R alter the internal dynamics of the GTP-bound active and GDP-bound inactive HRAS, particularly the switch domains. Figure 4: Concerted motions of structural domains from the GTP- and GDP-bound complexes. (**A**) GTP-bound WT, (**B**) GDP-bound WT, (**C**) GTP-bound A59E, (**D**) GDP-bound A59E, (**E**) GTP-bound K117R, and (**F**) GDP-bound K117R, respectively. Arrows on a structure represent the PC vectors. It is well known that the switch domains SW1 and SW2 are involved in the binding of HRAS to its effectors. Hence, the changes in dynamics behavior of the SW1 and SW2 caused by A59E and K117R affect the HRAS-effector binding and regulate the activity of HRAS, which is supported by the later distance analyses in the FELs. In addition, the two current mutations also disturb the dynamic behavior of the loop L3 and the helix \(\alpha\)3, which are involved in the allosteric position of HRAS. Therefore, A59E, and K117R certainly influence the allosteric regulation of HRAS activity.