Catalytically Competent Non-transforming H-RASG12P Mutant Provides Insight into Molecular Switch Function and GAP-independent GTPase Activity of RAS

RAS mutants have been extensively studied as they are associated with development of cancer; however, H-RASG12P mutant has remained untouched since it does not lead to transformation in the cell. To the best of our knowledge, this is the first study where structural/dynamical properties of H-RASG12P have been investigated -in comparison to H-RASWT, H-RASG12D, RAF-RBD-bound and GAP-bound H-RASWT- using molecular dynamics simulations (total of 9 μs). We observed remarkable differences in dynamics of Y32. Specifically, it is located far from the nucleotide binding pocket in the catalytically-active GAP-bound H-RASWT, whereas it makes close interaction with the nucleotide in signaling-active systems (H-RASG12D, KRAS4BG12D, RAF-RBD-bound H-RASWT) and H-RASWT. The accessibility of Y32 in wild type protein is achieved upon GAP binding. Interestingly; however, it is intrinsically accessible in H-RASG12P. Considering the fact that incomplete opening of Y32 is associated with cancer, we propose that Y32 can be targeted by means of small therapeutics that can displace it from the nucleotide binding site, thus introducing intrinsic GTPase activity to RAS mutants, which cannot bind to GAP. Therefore, mimicking properties of H-RASG12P in RAS-centered drug discovery studies has the potential of improving success rates since it acts as a molecular switch per se.

crystal structures of various G12 mutants of RAS subtypes [16][17][18][19][20][21][22][23] . On the other hand, the mechanistic insight could only be provided by means of molecular dynamics studies. Lu et al. have shown that G12 mutants of K-RAS4B such as G12C, G12D and G12V cause inactive-to-active conformational change by triggering different dynamics in RAS 8 . Compared to K-RAS4B G12C , K-RAS4B G12D and K-RAS4B G12V distort the nucleotide binding pocket more in the GTP-bound state, whereas K-RAS4B G12C causes larger conformational change in the GDP-bound state which leads to higher exposure of the nucleotide 8 . In another molecular dynamics study, G12D and G12V oncogenic mutants of H-and K-Ras have been comparatively studied and H-RAS G12V has been shown to be more flexible than its K-RAS counterpart. In the same study, it has been also shown that H-RAS G12V mutant adopted a conformational state that prevented protein from interacting with the effector 24 . Moreover, Vatansever et al. have shown that GTP-binding stabilizes motions in K-RAS and leads to residue correlations having long decay times 25 .
In spite of close association of RAS mutants with development of cancer, H-RAS G12P mutant does not lead to cancer in the cell (non-transforming mutant). Similar to transforming RAS mutants, H-RAS G12P mutant also cannot bind to GAP since pyrrolidine ring of P12 points outward and prevents interaction between RAS and GAP effector 8,16,[26][27][28][29] ; however, it can still hydrolyze GTP using its intrinsic GTPase activity. Structurally, transforming H-RAS G12D (PDB ID: 1AGP) 16 and non-transforming H-RAS G12P mutant (PDB ID: 1JAH) 17 are very similar as the backbone RMSD between them is measured as 0.44 Å and increases up only to 1.4 Å when considering the side chain atoms as well. Consequently, this suggests that non-transforming mutant displays different dynamics that provides intrinsic GTPase activity to the protein, hence can be used as a model system to understand molecular mechanism of GAP-independent GTP hydrolysis.
In this study, we performed comparative analysis of structural and dynamical properties among H-RAS G12D , H-RAS G12P and H-RAS WT by means of long atomistic molecular dynamics simulations (total of 9 μs). Trajectories of GAP-bound and RAF-RBD-bound H-RAS WT were used to represent the catalytically-and signaling-active states of the protein, respectively. We showed that non-transforming H-RAS G12P mutant displays remarkable differences in terms of dynamics of the system. Specifically, Switch I and II are more stable in the non-transforming mutant which leads to have a more compact nucleotide binding pocket than the transforming mutant. Considering that Switch II is responsible for interaction with nucleotide exchange factors (GEF) the stability of this region increases the strength of interaction between RAS and GEF, thus expediting the rate of nucleotide exchange as in agreement with experimental data 16 . Interestingly, Y32, which has been shown to stimulate GTPase activity of RAS 30 , displays different dynamics in signaling-and catalytically-active states. It is positioned closer to the nucleotide in the former and in wild type RAS, whereas it is located far from the nucleotide in the latter. Moreover, the accessibility of Y32 in wild type protein increased upon GAP-binding. On the other hand, Y32 samples both of these states in the GTP-bound non-transforming mutant per se, and the accessibility of the residue decreases in the GDP-bound state, which agrees with experimental data 30 . Considering i) the role of Y32 in intrinsic GTPase activity of RAS 31,32 , ii) the relation between incomplete opening of Y32 and the onset of cancer 33 , and iii) undraggibility of RAS [34][35][36] we propose that Y32 can be used as an alternative site that can be targeted by means of small therapeutic molecules to displace it from the nucleotide binding pocket, thus introducing intrinsic GTPase activity to transforming RAS mutants, which cannot bind to GAP.

Results
In this study, we carried out comparative analyses of local and global structural/dynamic properties of H-RAS WT /H-RAS G12D /H-RAS G12P (wild-type/transforming mutant/non-transforming mutant), GAP-bound and RAF-RBD-bound H-RAS WT by means of atomistic molecular dynamics simulations to provide mechanistic insight into transforming activities of these systems. For H-RAS WT and H-RAS G12P , we studied GDP-bound states as well. RAF-RBD-bound and GAP-bound systems were used to represent signaling-and catalytically-active states of the protein, respectively.
Comparison of root-mean-square fluctuation (RMSF) profiles reveals significant differences in residues with functional relevance. Comparative RMSF analysis of the systems shows remarkable differences in fluctuation patterns of mutant RAS proteins. Specifically, the mutation of glycine to proline residue increases stability more in H-RAS G12P than H-RAS G12D . In particular, T35 and G60 which coordinate γ-phosphate of the nucleotide, and Q61 that participates in the catalytic activity of RAS less fluctuate in H-RAS G12P , whereas they relatively more fluctuate in the transforming mutant. (Compare red and green in Fig. 1 and see Table S1). It is also interesting that G60 and Q61 residues are more mobile in wild-type protein than in H-RAS G12P mutant despite the fact that both systems do not cause transformation in the cell (See Table S1). Moreover, residues 62-69, which are known to mediate binding of RAS to nucleotide exchange factor, namely GEF 37 , also relatively less fluctuate in the non-transforming mutant compared to wild-type RAS (See Table S2), which might explain why H-RAS G12P has lower dissociation constant for GDP 16 . Consequently, tight binding between GEF and RAS might trigger rapid nucleotide exchange in the non-transforming mutant than wild type protein. In addition, these residues fluctuate more in GDP-bound H-RAS WT than in GDP-bound H-RAS G12P mutant suggesting that the nucleotide binding pocket might be intrinsically more stable in non-transforming mutant independent of the phosphorylation status of the nucleotide (See Fig. S1). Besides mutants, we also compared fluctuation patterns of H-RAS WT and GAP-bound H-RAS WT and showed that the presence of the effector further stabilizes T35, G60 and Q61 residues. Interestingly, however, Y32 residue highly fluctuates in H-RAS G12P mutant and in GAP-bound RAS than the other systems.
the catalytically important residues are positioned closer to the nucleotide in the non-transforming mutant independent of the phosphorylation state of the nucleotide. In a recent 31P NMR study 38,39 , GppNHp-bound H-RAS WT has shown to be found in a mixture of different conformational states, namely State1 (inactive state) and State2 (active state) in solution. The active state is described by complete interaction between T35, G60, and γ-phosphate of GTP. The inactive state is further grouped into three sub-states, namely, inactive state 1, 2 and 3. In the first one, interaction between nucleotide, T35 and G60 is completely lost, whereas in the second one, interaction between G60 and nucleotide is maintained but the one formed between T35 and the nucleotide is lost. The inactive state 3 is discovered in a computational study which was conducted by Nussinov et al., where interaction between γ-phosphate of GTP and T35 is maintained, but the one formed between G60 and the nucleotide is lost 8,40 .
In this part, we investigated conformational state of the nucleotide binding pocket of the systems using the same atom pair (T35-G60) given above. To do so, we calculated probability distributions of two atom-pairs distances between side chain oxygen of T35 and GTP Pβ atom; namely Distance 1, and between backbone amide of G60 and GTP Pβ atom; namely Distance 2. For comparison, we utilized corresponding distances in crystal structures of GppNHp-bound H-RAS WT (PDB ID:5P21) 41 complex, where Distance 1 and Distance 2 were measured as 5.5 Å and 6.2 Å, respectively. In general, the nucleotide is tightly coordinated by T35 and G60 residues in all of the systems, except H-RAS G12D , as evident from sampling relatively longer distance values in Distance 1 and 2 (See Fig. 2). Similar coordination is also observed for K-RAS4B G12D mutant 8,40 Fig. 3A), which corresponds to the active state with a compact nucleotide binding pocket. In addition, RAF-RBD-bound system resembles State 2, whereas GAP-bound system resembles a mixture of State 2 and Inactive State 3 (shorter Distance 1 and longer Distance 2 values), albeit with low probability, where interaction between Pβ atom of the nucleotide and backbone amide of G60 is lost but the one formed between T35 and the nucleotide is maintained (See Fig. 3B). We also investigated the impact of phosphorylation status of the nucleotide on the conformational state of the catalytic site in non-transforming mutant and H-RAS WT . To do so, we compared probability distributions for the same atom pairs as given in Fig. 2. We showed that exchange of GDP by GTP causes tight coordination of the nucleotide by both T35 and G60 which is evident by left shifts in Distance 1 & 2 values in both H-RAS G12P mutant and H-RAS WT (Compare A-C and B-D in Fig. 4), which is similar to what is observed for K-RAS4B G12D and K-RAS4B G12C mutants 40 . Moreover, T35 and G60 sample shorter distances from the nucleotide in both GDP-and GTP-bound H-RAS G12P mutant compared to H-RAS WT which might expedite the organization of the catalytically important residues around the nucleotide binding pocket.    Here, the electrostatic repulsion between negatively charged aspartic acid residue and GTP in H-RAS G12D mutant causes this residue to be repelled from the nucleotide binding site, thus leaving a space for Y32 to interact with GTP. On the other hand, the abovementioned hydrogen bond is not formed in the non-transforming mutant and GAP-bound H-RAS WT as it is evident from wider distance distributions which are measured between the side chain of Y32 and Pβ atom of GTP (See Fig. 6 (See A (red and yellow), B and E). Detailed analysis of the trajectory of non-transforming mutant showed that the proline residue at 12 th position in H-RAS G12P mutant is closely positioned to GTP, thus leaving partly no space for Y32 to interact with the nucleotide.
Considering the fact that RAF-RBD-bound H-RAS WT represents the signaling-active state of RAS, it is expected to observe that Y32 makes close interaction with the nucleotide, thus preventing access to GTP for hydrolysis. Similarly, Y32 is not accessible also in the transforming mutant, making close interaction with the GTP, thus preventing access to the nucleotide and locking RAS in the "on" state. On the other hand, Y32 is dominantly exposed in the catalytically-active GAP-bound H-RAS WT , which is also expected because, in this way, GTP can be accessed by the nucleophilic water, thus triggering hydrolysis of the nucleotide. Interestingly, in the non-transforming H-RAS, Y32 samples both states ("exposed" and "non-exposed"), albeit with low probability of the "non-exposed" state. However, Y32 is exposed in H-RAS WT upon GAP-binding as it is found in the "exposed" state in GAP-bound H-RAS WT but not in H-RAS WT trajectories. Moreover, we also showed that dynamics of Y32 in H-RAS G12P mutant is modulated by the phosphorylation status of the nucleotide as Y32 can be closely positioned to the nucleotide in GDP-bound RAS (See Fig. SI-2). Lastly, it is also important to emphasize that Y32 is positioned closer to the nucleotide in another mutant RAS subtype as well, namely, K-RAS4B G12D (PDB ID:4DSN) 18 as shown in Fig. 7 suggesting that the incomplete opening of Y32 is conserved among different mutant RAS subtypes.
Local dynamic properties also emerge in dominant global motions of systems. Beside exploring local structural/dynamics properties of the systems, we also investigated dominant collective motions in trajectories by means of essential dynamics analysis. Here, we depicted first three eigenvectors which cumulatively constitute 50% of the overall motion and highlight distinctions in dynamics of the systems as shown in Fig. 8. In general, we observed that local system-specific properties also emerge as dominant global motions in the systems, which are evident from fluctuation profiles obtained from projection of the trajectories along their first three essential eigenvectors (See Fig. 8A). According to that, Switch I and II regions dominate overall motion in transforming mutant as evidenced by higher fluctuation in these regions with respect to the nucleotide binding pocket. This is represented by extreme structures that are obtained by projection of trajectories along their first eigenvectors (See Fig. 8B). On the other hand, motion of Y32 contributes more to the overall motion in non-transforming mutant and GAP-bound H-RAS WT (See red and orange in Fig. 8A).

Discussion
To the best of our knowledge, this is the first study, where structural and dynamical properties of non-transforming H-RAS G12P mutant have been revealed. Our results showed that glycine to proline mutation introduced stability to H-RAS G12P as evident from lower flexibility of Switch I & II in this non-transforming mutant. Interestingly, despite the fact that both wild type protein and H-RAS G12P do not cause transformation, the latter resembles more catalytically competent state than the former. In particular, relatively lower fluctuation of the catalytically important residue Q61 in both GDP-and GTP-bound states of H-RAS G12P might expedite proper organization of the nucleotide binding pocket, thus increasing GTPase activity of non-transforming mutant compared to wild type protein, which agrees with experimental data 16 .
Apart from T35, G60 and Q61, we also observed remarkable differences in dynamics of Y32. Specifically, it is positioned closer to the nucleotide in signaling-active systems, which are represented by RAF-RBD-bound  www.nature.com/scientificreports www.nature.com/scientificreports/ H-RAS WT and H-RAS G12D , thus preventing accessibility of GTP for hydrolysis. On the other hand, it is located far from the nucleotide binding pocket in catalytically-active state, which is represented by GAP-bound H-RAS WT complex. Interestingly, however; Y32 samples both of these orientations in H-RAS G12P . Considering the fact that orientational dynamics of Y32 is crucial for proper function of RAS 30,33 , understanding simultaneous sampling of "exposed" and "non-exposed" states of Y32 can provide insight into intrinsic molecular switch function of H-RAS G12P . Presumably, when Y32 is positioned closer to and Q61 is positioned far from the nucleotide H-RAS G12P can conduct signal; however, when Y32 is exposed and Q61 is positioned closer to the nucleotide, non-transforming mutant catalyzes hydrolysis of GTP.
In light of these findings, we propose that Y32 can be used as an alternative site which can be targeted by small therapeutic molecules to displace it from the nucleotide binding pocket, thus introducing intrinsic GTPase activity to transforming RAS mutants that cannot bind to GAP. Moreover, we believe that mimicking structural and dynamical properties of H-RAS G12P will improve success rates in drug discovery studies since it resembles catalytically competent state more than H-RAS WT .  44 . Corresponding crystal structures were retrieved from Protein Data Bank (PDB). In order to prepare GTP and GDP-bound H-RAS G12P systems, phosphomethylphosphonic acid guanylate ester (GCP) was converted to GTP and GDP by substituting C 3 B with O and removing γ-phosphate, sequentially. For H-RAS G12D and H-RAS WT , conversion of phosphoaminophosphonic acid-guanylate ester (GNP) to GTP was done by interchanging N 3 B with O. As a side note, GDP bound H-RAS WT does not require any manipulation since it has been resolved with GDP. For GAP-bound H-RAS WT , aluminum fluoride (AlF 3 ) was extracted and GDP was exchanged with GTP. For RAF-bound H-RAS WT system, acetate ion, (2R,3S) -1,4-dimercaptobutane-2.3-diol, and calcium ion were removed from the system, and GNP was substituted with GTP as it has done for both H-RAS G12D and H-RAS WT . Lastly, GTP-bound K-RAS4B G12D were prepared by removing 1,2-ethanediol and substituting GCP with GTP in the same way mentioned for H-RAS G12P . Also, crystal waters which are located within 5 Å of nucleotide were kept for all the systems. After these manipulations, protonation states of amino acids for all systems were assigned by using PropKa server 45 at pH 7.4. By taking periodic boundary conditions (PBCs) into account, thickness of water layer was adjusted as 9 Å, except GAP-and RAF-RBD-bound H-RAS WT . For these systems, it was set as 11 Å and 13 Å, respectively. Furthermore, TIP3P 46 water model was utilized, and systems were neutralized with NaCl.  www.nature.com/scientificreports www.nature.com/scientificreports/ for 4.8 picoseconds (ps) both in NVT and NPT ensembles. Temperature and pressure were set 310 K and 1 atm, respectively. Time step was adjusted to 2 femtoseconds (fs) in order to capture the fastest motions within systems. Cut off value for non-bonded interactions was set to 12 Å and long-range electrostatic interactions were computed by using particle mesh Ewald (PME). Furthermore, trajectory snapshots were saved in every 5 ps. Both GDP/GTP-bound H-RAS WT and H-RAS G12P were simulated for total of 4.8 μs, whereas GTP-bound H-RAS G12D , RAF-RBD-bound and GAP-bound H-RAS WT were performed in total 3.6 μs. Lastly, MD simulation of GTP-bound K-RAS4B G12D was performed for 0.6 μs. Here, we performed two separate simulations for the systems, each of which was started with different initial velocity. The results were presented as a combination of these two replicas.

Methods
Root-mean-square fluctuation (RMSF). Root-mean-square fluctuation was computed as following: where C ij corresponds to covariance matrix. A change in position from time-averaged structure for each coordinates of all atoms i and j was denoted as M ij Δr i Δr j . Covariance matrices were generated as shown in above. δ = v Cv 2 Diagonalisation of covariance matrices provided a set of eigenvalues and eigenvectors δ 2 , and v, respectively. Computation and diagonalisation of covariance matrices were done by making use of 'gmx covar' module of GROMACS while 'gmx anaeig' module of GROMACS was used to obtain eigenvectors and eigenvalues from diagonalized covariance matrices 49 .