# GTP-Bound N-Ras Conformational States and Substrates Are Modulated by Membrane and Point Mutation
## 2 Results  
The first set of MD simulations was performed for N-Ras (see Figure 1) wild-type (WT) in water with salt at physiological concentration, without membrane (see Figure 2A). Since most experimental and modeling studies on conformational state definitions [38; 41; 43] were conducted in similar conditions, we used these simulations as a reference for characterizing state 1 and state 2 (and their corresponding substates) at the molecular level.  
Figure 1: **N-Ras protein.** (**A**) The tertiary structure of the N-Ras protein. The five \(\alpha\)-helices are colored in different colors, \(\beta\)-sheets are in yellow, and loops are in gray. (**B**) The functionally important regions, switch I (red) and switch II (blue), the phosphate-binding P-loop (green), and the guanosine triphosphate (GTP) molecule (atom-type-colored licorice representation). (**C**) The two initial configurations used for N-Ras were at \(\sim\)45\({}^{\circ}\) with the membrane (bottom left) and quasi-parallel to the membrane (bottom right). (Structure color-coding, as in (**A**), GTP representation and color-coding as in (**B**).) Cysteine palmitoyl and farnesyl lipid modifications (PAL and FAR, respectively, shown in orange licorice representation) anchor N-Ras to the membrane model (in gray, with P atoms in bead representation).  
**State 1 \"inactive\" state.** We used the relatively loose characterization of [PERSON] et al. for the \"inactive\" state 1 at the molecular level, i.e., the conformation where the Thr35  
Figure 2: **Time evolution of representative simulation trajectories and the conformational distribution of (sub)states of N-Ras, as calculated over all the performed simulations.****(A) In solution, without membrane: time evolution for WT (a,b) and G12V (c), and distribution of (sub)states (d): (B) Inserted in/bound to model membrane: time evolution for WT (a) and G12V (b,c), and distribution of (sub)states (d). Time evolution plots represent the system dynamics, characterized by specific distances, involving the following atoms: amino acid backbone oxygen (O), \(\gamma\) oxygen of GTP (O\({}_{\gamma}\)), terminal oxygen at the end of Tyr ring (OH), sidechain oxygen of Thr (OG), magnesium (Mg). Subscript numbers indicate the residue to which the atoms belong. The distances are color-coded as follows: (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.**hydrogen bond to Mg\({}^{2+}\) ion is broken [41] (i.e., the violet line values in the trajectories increase dramatically Figure 2).  
**State 2 exhibits three substates.** This is the \"active\" state, in which Thr35 binding to Mg\({}^{2+}\) and GTP is crucial [38]. State 2 was shown to exhibit two substates for H-Ras, corresponding to two different functions [43]. Their major conformational difference involves the different orientations of Tyr32: \(OX\) and \(OZ\)[43]. In our simulations, we found that N-Ras exhibits, besides the above-mentioned orientations, a third _novel_ orientation of Tyr32, \(OY\), toward Ala18. All three have quasi-perpendicular orientations, as shown in Figure 3. Substate 2\({}_{OX}\) features the Tyr32 sidechain oriented toward Gly12 and, therefore, toward the GTP-binding pocket; substate 2\({}_{OY}\) has the Tyr32 sidechain oriented parallel to this pocket, toward Ala18, while substate 2\({}_{OZ}\) has the Tyr32 sidechain oriented toward the bulk water (perpendicular to the plane formed by the other two directions). In the latter state, Tyr32 corresponds to its position when GAP is bound to Ras (PDB id 1 WQI [53]).