# GTP-Bound N-Ras Conformational States and Substrates Are Modulated by Membrane and Point Mutation
## 1 Introduction  
Ras proteins are well-studied small GTPases that function as molecular switches between GTP-bound active and GDP-bound \"inactive\" forms to mediate signal transduction pathways that regulate cell growth, differentiation, and proliferation [1, 2]. Now it is known that Ras proteins are post-translationally modified membrane-bound proteins that form non-overlapping, dynamic, nano-sized domains (nanoclusters) in an activation state-/isoform-dependent manner [3, 4]. These nanoclusters act as binary switches [3, 5, 6], whose activation is aided by guanosine exchange factors (GEFs), which stimulate the dissociation of GDP and subsequent binding of GTP (GTP is \(\sim\)9-fold more abundant in the cytosol than GDP [7]). GTP-bound Ras activates a great variety of downstream signaling pathways by interacting with many effectors [8, 9]. Ras signaling is terminated by the hydrolysis of the bound GTP, which can be accelerated by several orders of magnitude by GTPase-activating proteins (GAPs) [10]. Oncogenic point mutations are resistant to GAP binding and, hence, render them constitutively active. Most importantly, these mutations are associated with \(\sim\)30% of all cancers, and in some specific cancers, they are found in over 90% of cases [11, 12], as well as developmental disorders [13, 14]. Nonetheless, little success has been achieved thus far in developing clinically effective targeted therapy using oncogenic point mutation sites [15]. H-, N-, and K-Ras are three isomers that are ubiquitously expressed in humans. While they share nearly identical catalytic machinery (G-domain variations are less than 15%), their C-terminal hypervariable region (HVR), which contains the lipid modifications (lipidanchor), is \(\sim\)85% different. The mechanism by which the membrane-binding motif attaches to cellular or model membranes was extensively investigated both experimentally (reviews in Refs. [16; 17]) and computationally [18; 19; 20; 21; 22; 23; 24; 25]. Nonetheless, this knowledge alone is not enough to explain the complexity of the spatial and temporal organization of the three isomers into non-overlapping nanoclusters on the plasma membrane surface [3; 6; 26]. Further experiments on full-length proteins [27; 28; 29; 30; 31], as well as on simplified model peptides [32; 33; 34; 35], have led to the consensus that H-Ras prefers liquid-ordered domains, N-Ras prefers the border between the liquid-ordered (\(L_{o}\)) and liquid-disordered (\(L_{d}\)) domains, while K-Ras prefers the \(L_{d}\) domain. Computer simulations have shed light on the role and importance of lipid anchors [36] in the localization of the isomers, and have highlighted the effects of peptide concentration and lipid composition on the formation and domain partitioning of the anchor peptides into nanoclusters [37]. Extensive experimental structural studies on very similar isomers have led to an impressive database of Ras structures (predominantly H- and K-Ras). However, all these crystallographic structures show that Ras proteins exhibit multiple conformational states, mainly driven by the so-called switch I and switch II regions. Active (GTP-bound) conformations can be grouped into two main states at dynamic equilibrium: the \"inactive\" state 1 and \"active\" state 2 [38]. State 1 interacts with GEFs and, therefore, is characterized by an increased protein surface area (due to the opening in the nucleotide-binding pocket, required for the interaction with GEFs) [39], which, in turn, leads to higher switch I flexibility [40]. At the molecular level, the major characteristic of state 1 was shown to be the broken hydrogen bond between Thr35 and the Mg\({}^{2+}\) ion [41]. State 2, on the other hand, can interact with a variety of effectors [39; 42]. Recently, [PERSON] et al. [43] proposed that, at least for H-Ras, state 2 is in fact split into two separate conformational substates, corresponding to the Ras-effector interaction, and Ras-GAP interaction, respectively. The structural discrepancies between the two substates are minor.