# GTP-Bound N-Ras Conformational States and Substrates Are Modulated by Membrane and Point Mutation
## 3 Discussion  
Overall, our findings suggest that the conformational dynamics of Ras proteins, specifically N-Ras, play a critical role in their oncogenicity and interaction with the cell membrane. The crystallographic structures of Ras proteins have revealed the existence of multiple conformational states, indicating the flexibility of these proteins. Among the GTP-bound states, two main states have been identified: the \"inactive\" state 1 and the \"active\" state 2. Recent studies on H-Ras have shown that state 2 can exist in two substates, distinguished by the orientation of Tyr32 toward the GTP-bound pocket or outwards [43]. In this study, we investigated the conformational dynamics of N-Ras and discovered an additional substate of state 2. This substate is characterized by the orientation of Tyr32 toward Ala18 and in parallel to the GTP-bound pocket.  
Interestingly, we found that this substate is highly sampled in the G12V mutation of N-Ras in the membrane, which is commonly associated with oncogenicity, but barely present in the wild-type form. Moreover, our molecular dynamics simulations showed that the membrane-binding of N-Ras prevents the sampling of substate \(2_{OY}\) and favors the sampling of substate \(2_{OX}\). Therefore, we believe that in WT, the membrane's role is also  
\begin{table}
\begin{tabular}{l c c c c c} \hline \hline \multirow{2}{*}{**Location**} & \multirow{2}{*}{**Type**} & \multicolumn{4}{c}{**Avg. Number of Na\({}^{+}\) Ions and Std. Dev.**} \\ \cline{3-6}  & & **State 1** & **State 2\({}_{OX}\)** & **State 2\({}_{OY}\)** & **State 2\({}_{OX}\)** \\ \hline
**A.** & **WT** & 2.53 \(\pm\) 1.52 & 0.56 \(\pm\) 0.58 & 0.40 \(\pm\) 0.61 & 0.15 \(\pm\) 0.36 \\
**sol. only** & **G12V** & 1.56 \(\pm\) 0.85 & - & - & - \\ \hline
**B.** & **WT** & 1.72 \(\pm\) 0.58 & 0.42 \(\pm\) 0.52 & 0.75 \(\pm\) 0.58 & 0.06 \(\pm\) 0.24 \\
**membr-bound** & **G12V** & 2.30 \(\pm\) 0.82 & 1.23 \(\pm\) 0.65 & 0.88 \(\pm\) 0.55 & - \\ \hline \hline \end{tabular}
\end{table}
Table 1: The table contains the average number of Na\({}^{+}\) ions (and their corresponding standard deviations) found in the vicinity (within 4.5 Å) of N-Ras\({}^{\prime}\) GTP molecule for each of the four (sub)states (state 1 and state 2, with its three substates—OX, OY, OZ).  
to evade this specific substate \(2_{OY}\), and enhance N-Ras' transition into the predominant substate \(2_{OX}\), previously identified as playing a key role in the conformational transition to the conformation substate \(2_{OZ}\) (interacting with GAP's arginine finger) [43].  
The G12V mutation, on the other hand, has the opposite effect, minimizing substate \(2_{OX}\) sampling and drastically increasing the sampling of substate \(2_{OY}\), rendering this mutation oncogenic. Interestingly, we observed that the G12V mutation in the absence of the membrane hinders the sampling of the substate related to the binding of the GTPase-activating protein (GAP) (substate \(2_{OZ}\)), and remains in state 1 until the end of our simulations.  
In conclusion, our study reveals that the conformationally \"active\" state 2 of Ras exhibits three distinct substates, and highlights the significance of conformational dynamics and the influence of the cell membrane in Ras protein function and oncogenicity. Understanding the conformational landscape and membrane interactions of Ras proteins may provide valuable insights for the development of targeted therapies against Ras-driven cancers.