## 1 Introduction
The colors of the surfaces are indicated in red and blue. Structures of GTP **(C)** and GDP **(D)** are characterized by stick modes and colored according to their elements. work analysis, which suggested that the R135K mutation triggers the allosteric unbinding of monobody from HRAS [56]. [PERSON] et al. employed MD simulations and PCA to quantitatively decipher the structural and dynamical features of the active and inactive states of GTP- and GDP-bound HRAS. Their results indicated that the inactive-to-active transformation of HRAS is a multiphase process involving the rearrangement of the switches SW1 and SW2 [57]. Furthermore, different research groups utilized accelerated molecular dynamics (aMD) simulations [58] to study the conformational changes in RAS proteins mediated by mutations [59; 60]. [PERSON] et al. employed GaMD simulations and FELs analysis to explore the alterations in the switch states of HRAS induced by point mutations D33E, A59T, and L120A and to clarify the impact of these mutations on the conformational dynamics of HRAS [61]. Although those experimental and computational studies provided valuable insights into the regulation of RAS activity, a comprehensive understanding of the conformational dynamics of HRAS and its function is still lacking. Therefore, further investigations are essential to exploring the conformational changes of HRAS caused by mutations to deepen our understanding of the function of HRAS. To further investigate the molecular mechanism underlying conformational changes in HRAS caused by point mutations, two specific mutations, A59E [33] and K117R [27], were selected for analysis in this study. The mutated sites are illustrated in Figure 1B. The A59E mutation occurs near the SW2 region, while the K117R mutation is at the allosteric site. The substitution of A59 with E59 not only leads to an elongation of the sidechain, but also introduces a negative charge. On the other hand, the K117R mutation only alters the geometry of the sidechain. Compared to the frequently detected mutations of codons 12 and 13, A59E and K117R also lead to functional differences in HRAS. The study by [PERSON] et al. proposed that the K117R mutation disrupts guanine nucleotide binding and has similar functional implications as mutations affecting GTP hydrolysis and causing human diseases [27]. On the other hand, A59E is a mutation of RAS that is associated with cancer. The research conducted by [PERSON] et al. confirmed that both A59E and phosphorylation significantly accelerate the intrinsic exchange [33]. In addition, K117R has weaker effects on downstream c-Jun N-terminal kinase signaling. Thus, to explore the effects of these two mutations on the conformational changes in HRAS, we constructed six systems: wild-type (WT) HRAS bound to GDP, A59E HRAS bound to GDP, K117R HRAS bound to GDP, WT HRAS bound to GTP, A59E HRAS bound to GTP, and K117R HRAS bound to GTP. The structures of GDP and GTP are depicted in Figure 1C,D, respectively. As reported in previous studies, GaMD simulations have demonstrated great success in exploring the dynamics of target conformations [62; 63; 64; 65; 66; 15; 67]. Therefore, GaMD simulations were used in this study to enhance the conformational sampling of HRAS. Subsequently, post-processing analyses were performed on the GaMD trajectories, including principal component analysis (PCA), dynamics cross-correlation maps (DCCMs) [68; 69; 70; 71], and free energy landscape (FEL) analyses. This work is anticipated to provide valuable insights and information to aid in future drug design targeting RAS proteins.