# GTP-Bound N-Ras Conformational States and Substrates Are Modulated by Membrane and Point Mutation
## 2 Results
### Characterization of the Conformational (Sub)States  
To monitor how substates shift, one approach is to investigate the conformational changes in the residues surrounding the specific region of interest during the simulation. This helps in understanding how the dynamics of these residues may influence the overall behavior of the system. Figure 2 depicts the distances between (i) O\({}_{12}\)-O\({}_{\gamma}\) in blue, (ii) OH\({}_{32}\)-O\({}_{18}\) in green, (iii) OG\({}_{35}\)-Mg in violet, (iv) OH\({}_{32}\)-O\({}_{\gamma}\) in red, and (v) OH\({}_{32}\)-OH\({}_{40}\) in yellow. By inspecting the changes in these distances, one can gain insights into the structural rearrangements that occur in the system. The distances between the OG\({}_{35}\) and Mg in all substates of state 2, as shown by the violet line in Figure 2, are very similar, measuring approximately 2.0 A. These values closely match the measurements obtained from the crystal structures of state 2, such as the substate 2\({}_{OX}\) with the PDB identification 3K8Y [54]. This suggests that the changes in the OG\({}_{35}\)-Mg distance within state 2 are consistently maintained across different substates. In contrast, state 1 is distinguished by the breaking of the hydrogen bond between the Mg\({}^{2+}\) ion and Thr35. Consequently, there is a significant increase in the distance between OG\({}_{35}\) and Mg, a change that has been observed in the crystal structure of state 1, as evidenced by the 4 EFL crystal structure [41] (see Figure 4C). The identification of this alteration in the OG\({}_{35}\)-Mg distance serves to further distinguish state 1 from state 2 and highlights the distinct molecular characteristics of each state. The distance between the O\({}_{12}\)-O\({}_{\gamma}\) of GTP remains constant at around 5.3 A in all the substates of state 2 of the WT systems. This value is relatively close to the distance observed  
Figure 3: **N-Ras exhibits three substates of state 2. ** These are mainly characterized by the quasi-perpendicular orientations of Tyr32. Protein is shown in the secondary structure representation in gray, GTP in the atom-type coded colors. in the crystal structure of the substate 2\({}_{OX}\) of H-Ras (3K8Y [54]). However, when the G12V mutation is present, this distance is shifted, increasing to values above 8 A. This shift is attributed to the significantly larger size of the Val12 residue compared to the wild-type Gly12 residue (see Figure 4A). In substate 2\({}_{OX}\), the OH\({}_{32}\)-O\({}_{\gamma}\) distance fluctuates around 2.7 A. On the other hand, substate 2\({}_{OZ}\) displays larger fluctuations in the OH\({}_{32}\)-O\({}_{\gamma}\) distance, which centers around 7.5 A. These fluctuations are due to the repositioning of the Tyr32 residue toward the bulk of the protein. This repositioning could be connected to the hydrolysis process associated with substate 2\({}_{OZ}\)[43]. The newly identified substate 2\({}_{OY}\)'s orientation leads to the farthest positioning of the Tyr32's terminal ring oxygen atom (OH) from the \(\gamma\) phosphate group of GTP, resulting in an OH\({}_{32}\)-O\({}_{\gamma}\) distance of approximately 11.3 A. In state 1, the switch I loop moves away from the GTP, and the breaking of the Thr35-Mg hydrogen bond causes a significant increase in the OH\({}_{32}\)-O\({}_{\gamma}\) distance as compared to other substates. Overall, these findings highlight the dynamic nature of the OH\({}_{32}\)-O\({}_{\gamma}\) distance and its crucial role in characterizing all the (sub)states of Ras proteins. The conformation of substate 2\({}_{OY}\) is unique due to the specific positioning of the Tyr32 residue toward Ala18. This particular orientation is characterized by the remarkably short OH\({}_{32}\)-O\({}_{18}\) distance, measuring only 4.3 A. In sharp contrast, the other substates exhibit significantly larger distances, exceeding 12 A (see Figure 4B). To closely monitor the changes of switch I, we calculated the distance OH\({}_{32}\)-OH\({}_{40}\). In state 1, our simulations show a wide range of values.