# GTP-Bound N-Ras Conformational States and Substrates Are Modulated by Membrane and Point Mutation
## 2 Results
### Characterization of the Conformational (Sub)States
However, the repositioning of Tyr40 close to Tyr32 (the OH\({}_{32}\)-OH\({}_{40}\) distance value becomes significantly smaller) leads to the destabilization of Thr35 and determines the bond between the magnesium ion and the side chain oxygen of Thr35 to rupture. This change promotes the protein's conversion from substate 2\({}_{OX}\) into state 1, as depicted in the 4 EFL [41] crystal structure (OH\({}_{32}\)-OH\({}_{40}\) distance around 4.4 A). The simulations do not show any important changes in switch I for the \(OX\) and \(OZ\) substates (the distance value is around 11.6 A). Figure 4: **Overlap of N-Ras structures in different (sub)states. ** Protein G-domain secondary structure in transparent cartoon representation, Tyr32 and Gly/Val12 in stick-and-balls representation, Thr35 in licorice representation and Ala18 in balls representation are colored in the first color in parannhesis for each subfigure, while GTP in licorice representation is colored in the second listed color. (**A**). Substate 2\({}_{OX}\) in membrane WT (dark blue/blue) and G12V (cyan/light blue) together with the 5 UHV N-Ras crystal structure (pink/red) [52]. (**B**). Substate 2\({}_{OY}\) in membrane WT (green/dark green) and G12V (light green/white) together with substate 2\({}_{OZ}\) (red/light red). Ala18, shown only in this subfigure, highlights the different orientations of Tyr32 in the two substates. (**C**). State 1 in membrane WT (white/gray) and G12V (pink/mauve) together with the 4 EFL H-Ras state 1 crystallographic structure (yellow/orange) [41]. Thr35 is shown instead of Tyr32 in this subfigure, to highlight its departure from the GTP-bound Mg\({}^{2+}\) ions in state 1. From a different perspective, we also focused our attention on characterizing the membrane-bound protein orientation with respect to the stretch of the HVR domain using two reaction coordinates (RCs) equivalent to those employed on G12V K-Ras [55]: (1) the angle between a vector along the \(\beta\)1 sheet (residues 2-5) and the membrane normal (angle), and (2) the distance between C\({}_{\alpha}\) atoms of E132 on the \(\alpha\)4 helix and L184 on the HVR domain (dist). From the contour plots based on these two RCs (see Figure S7), one can observe that G12V mutations are accompanied by significant deviations or changes from the inherent dynamics characterized by these two RCs. For WT, the substates OY and OZ of state 2 exhibit a more specific positioning of the G domain orientation with respect to a fairly compact HVR. Substate \(2_{OX}\) overlaps with the other two but also exhibits a medium-stretched (and specific) HVR and a conformational connection \"pathway\" toward a very narrow HVR and specific orientation of state 1, consistent with state 1 acting as a \"pool\" for the WT active state 2 [43; 56]. However, with the G12V mutation, these RCs become less specific for state 1 and substate \(2_{OY}\), with wide ranges of G-domain orientations and HVR distances being sampled. Substate \(2_{OX}\) is not sampled, and in substate \(2_{OX}\), the conformations sampled become selective for a medium-stretched HVR and a G domain orientation parallel to the membrane. Hence, the conformational overlap sampled in our simulations between state 1 and substate \(2_{OX}\) becomes practically nonexistent. These findings suggest that the G12V mutation has an impact on the orientation of the G domain with respect to the membrane, thereby altering the protein's interaction with the membrane.