# GTP-Bound N-Ras Conformational States and Substrates Are Modulated by Membrane and Point Mutation
## 1 Introduction
The main distinction is characterized only by the different orientations of the Tyr32 residue: toward the GTP pocket (Tyr32\({}_{in}\)) and toward the bulk solution (Tyr32\({}_{out}\)). The latter orientation is in agreement with Tyr32's position in the crystallographic structure of Ras-GAP structures, required for the GAP Arg finger insertion needed for enhanced hydrolysis. In fact, not only are the sampled GDP/GTP-bound states different, but their substates are also membrane-modulated and dependent on catalytic domain orientation [22; 44; 45; 42]. The existence of additional substates has been further investigated in recent years. Hence, [PERSON] et al. revealed that the K-Ras-GDP\(\cdot\)Mg\({}^{2+}\) product state has multiple stable substates in solution, suggesting that complexation with GEFs may involve a conformation-selection mechanism [46]. The use of pressure-induced crystallo-phase transitions provided a unique opportunity to investigate the structural determinants involved in the switching between Ras allosteric substates, without the need for mutations or external partners [47]. Since distinguishing between \"active\" and \"inactive\" molecular conformations is still very challenging, novel computational approaches have addressed this by using novel classification methods [48], free energy approaches [46], or density-based machine learning algorithms to cluster switch I and switch II loops into novel conformational subsets [49]. While a great amount of work has focused on H- and K-Ras, too little has been directed toward the study of N-Ras structural and conformational characteristics, although oncogenic N-Ras is the major cause of malignant melanomas, thyroid carcinomas, and some types of leukemia [12; 50], with Gly12 being one of its most important oncogenic mutations [12]. Hence, an order of magnitude fewer crystallographic structures are available for N-Ras (e.g., GDP-bound N-Ras (3 CON) [51] and GTP-equivalent GppNHp-bound N-Ras (5 UHV) [52]). While it has been shown that the Ras anchor plays a major role in localization and, hence, differentiation of the Ras isomers [36; 37], most of the work on structural Ras has focused on solvated Ras. Nonetheless, previous work has identified two membrane-bound catalytic domain Ras orientations (one parallel and one at an angle) [22], both nucleotide state- and isomeric-dependent [22; 44; 45]. Moreover, the G12V mutation was shown to adopt the parallel orientation for H-Ras [22]. However, it is still not clear how these findings can be applied to N-Ras. Hence, in this paper, we shift our focus to N-Ras and, by means of molecular dynamics simulations, we show that _GTP-bound N-Ras_ (i) exhibits three, Tyr32 orientation-characterized, conformational substates of state 2--the novel substate, has a quasi-parallel orientation of Tyr32 to the GTP-bound pocket, toward Ala18 (Tyr3\({}_{2 parallel}\)); (ii) membrane anchoring to a plasma membrane model provides increased stability of the N-Ras system and, therefore, the accuracy of the distribution of conformational states and substates (state 1 and three substates of state 2) of N-Ras is strongly enhanced by the membrane presence; (iii) the Tyr3\({}_{2 parallel}\) substate is barely sampled by the membrane-bound wild-type N-Ras; (iv) G12V mutation inhibits the GAP-bonded corresponding substate of state 2 (Tyr3\({}_{2 out}\)), which is required for enhanced hydrolysis of GTP, rendering it oncogenic.