# GTP-Bound N-Ras Conformational States and Substrates Are Modulated by Membrane and Point Mutation
## 2 Results
### Dynamics and Distribution of (Sub)States  
The gray bars in Figure 2A(d) show that state 1 is the most populated state (\(\sim\)40%), while substate \(2_{OX}\) and substate \(2_{OY}\) correspond to \(\sim\)30% and \(\sim\)20%, respectively. This is consistent with the recent attribution of state 1 as a \"pool\" for the \"active\" state 2 in the wild-type Ras [43; 56]. Although substate \(2_{OZ}\) is present only \(\sim\)3% of the time, it is crucial in the hydrolysis process, as it corresponds to the GAP-binding conformation [43]. Apart from the sidechain orientation, the three substates of state 2 can also be quantitatively distinguished by their root mean square deviation (RMSD) values with respect to the Ras crystal structure identified to be in substate \(2_{OX}\) (3K8Y [54]) (see Figure S1). The RMSD plot also shows the sharp transitions between the substates, when they occur directly (details are in the Supplementary Material). The overlay of the average structures for state 1 and state 2 shows significant variation at the loop of the SI region (see Figure S2). However, the core structure of the N-Ras protein remains very stable in all simulations, both in solution and membrane-bound, with C\({}_{\alpha}\) root mean square deviation (RMSD) less than 1.4 A from the corresponding crystal structure (Figure S3) and averages from 0.94 Ato 1.15 A. Nevertheless, significant differences are noted at switch I (SI) and switch II (SII) regions (see Figure S4), with mean RMSD values of 3.78 A-6.77 A and 4.27 A-5.64 A, respectively. These deviations are reflected in the C\({}_{\alpha}\) root mean square fluctuation (RMSF) calculated for each residue, and time-weight averaged over several trajectories for each (sub)state (see Figure 5). State 1 is characterized by overall larger fluctuations of both switches than state 2 in general, and larger fluctuations of SI compared to SII, unlike state 2. The RMSF plots for WT in solution only (black line in Figure 5) show some small differences between the substates of state 2 (see Figure S5), consisting of (i) the lowest fluctuations for SI and SII regions of substate \(2_{OZ}\) (which is consistent with the GAP-binding state corresponding structure), and higher on residues 105-107; and (ii) fluctuations for the SII region of substate \(2_{OY}\) comparable to those of state 1 (i.e., 50-100%, larger than on the other two substates of state 2), but only on residues 60-69. The PCA and cross-correlation analysis allowed us to identify the key motions and interactions within N-Ras (see Figures 6 and S6). The motions of switch I and switch II play a crucial role in the conformational changes of the N-Ras protein. The anti-correlated motion of these switch regions with other residues in the catalytic domain suggests that these regions may undergo opposite movements to facilitate the transition between different states. On the other hand, the reduced correlation observed in the active state suggests that other regions of the protein may become more rigid during activation (see Figure S6). Figure S6 shows the cross-correlation matrices (obtained using the NMWiz plugin [57]) of residue pairs in the N-Ras protein for the three substates of state 2: substate \(2_{OX}\), substate \(2_{OY}\), and substate \(2_{OX}\). We observed considerable correlations between residue pairs in substate \(2_{OX}\) and substate \(2_{OY}\). The two switch regions, SI and SII, show stronger anti-correlated motion with other residues of the protein catalytic domain. In contrast, in the active substate of N-Ras (substate \(2_{OX}\)), most cross-correlation values of residue pairs are close to 0, indicating a reduced correlation of these residue pairs. To verify which motions could account for RMSD variations, we performed a (PCA) from the MD trajectory. Results for the first 10 eigenvectors confirm that the greatest motions were contained within the first 3 eigenvectors and they indeed came from Switch I and Switch II (see Figure 6).