Author response: Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins. Editor's evaluation This study employs extensive MD simulations to probe the effect of phosphorylation of a tyrosine residue on the conformational ensemble of Ras GTPase. The insights form the basis for a screen of small molecule(s) that disrupt interaction with its target Raf kinase, and predictions are tested experimentally. Overall, the integrated approach is of interest to a wide range of biochemists and protein scientists and could potentially be used to modulate the activities of other proteins. https://doi.org/10.7554/eLife.79747.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The RAS gene family translates into four proteins, namely HRAS, NRAS, KRAS4A, and KRAS4B, that control mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and Ras-like (RAL) pathways (Barbacid, 1987; Malumbres and Barbacid, 2003; Lu et al., 2016a; Khan et al., 2019; Duffy and Crown, 2021; Simanshu et al., 2017; Ferro and Trabalzini, 2010; De Luca et al., 2012; Young et al., 2013; Knight and Irving, 2014). These small G proteins act as a binary switch as the activation of the protein is modulated by two types of nucleotides, namely, guanosine-triphosphate (GTP) and guanosine-diphosphate (GDP). The exchange of GDP for GTP is maintained by guanine exchange factors (GEFs) which, in turn, activates the RAS protein (Downward, 1990; Grand and Owen, 1991; Bourne et al., 1991; Wittinghofer and Pai, 1991; Lowy et al., 1991; Wittinghofer and Vetter, 2011; Takai et al., 2001; Lamontanara et al., 2014; Vetter and Wittinghofer, 2001; Lu et al., 2016a). Consequently, activated RAS proteins can interact with their downstream effectors, thus initiating cellular signaling pathways (Vetter and Wittinghofer, 2001; Cherfils and Zeghouf, 2013; Geyer and Wittinghofer, 1997; Lu et al., 2016a). Unlike GEFs, GTPase-activating-proteins (GAPs) accelerate the intrinsic GTPase activity of RAS, which provides a control mechanism for precise termination of respective signaling pathways (Wittinghofer et al., 1997; Lu et al., 2016a). RAS proteins are made up of two domains, namely, G domain (residues 1–172) and hypervariable region (173–188 or –189) (O’Bryan, 2019; Khan et al., 2020, Figure 1A). The G domain consists of effector (residues 1–86) and allosteric lobes (residues 87–172). The former, which is the invariant region, harbors the P-loop (residues 10–17), Switch I (30-38), and Switch II (59-76) regions, the last two of which adopt different conformational states depending on the type of the nucleotide (Wang et al., 2021). In particular, Switch I/II can be found in either open or closed conformation, both of which are described depending on the position of the domain with respect to the nucleotide-binding pocket. In the open conformation, Switch I/II is far from the nucleotide-binding pocket, whereas it is closer in the closed conformation. Importantly, the former prevents effector binding while the latter favors it. Moreover, it has been also shown that Switch II becomes less stable upon effector binding, which presumably allows RAS to cycle between catalytically incompetent and competent states in a timely manner that is important for maintaining the cell homeostasis (Johnson and Mattos, 2013; Khan et al., 2020). Figure 1 Download asset Open asset RAS phosphorylation/dephosphorylation cycle. (a) Important residues/regions that play pivotal role in RAS function are shown. (b) A schematic that illustrates the impact of tyrosyl phosphorylation on the GTPase cycle of HRAS. The tyrosyl phosphorylation at the 32nd position, which is mediated by Src kinase, causes impairment of RAF binding, thus terminating RAF/MEK/ERK signaling pathway as long as the phosphoryl group of Y32 is not detached by SHP2 Bunda et al., 2014. Since RAS proteins are involved in signaling pathways, which are responsible for cell growth, differentiation, and proliferation, mutations, which are frequently found at the 12th, 13th, and 61st residues (Prior et al., 2020; Prior et al., 2012, cause several cancer types Holderfield et al., 2014; Eser et al., 2014; Prior et al., 2012; Stephen et al., 2014; McCormick, 2015a; McCormick, 2015b; Krens et al., 2010; Lu et al., 2016a as a result of attenuated GTP hydrolysis and increased nucleotide exchange rate Vigil et al., 2010). For instance, HRASG12D was shown to be the dominant mutant in ductal carcinoma (Myers et al., 2016) caused resistance to erlotinib, which is used as an epidermal growth factor receptor tyrosine kinase inhibitor (Hah et al., 2014), in head and neck squamous carcinoma. As such, RAS proteins have been standing as hot targets in drug discovery studies which are focused on the development of therapeutics against cancer. In spite of extensive efforts that have been made to develop RAS inhibitors, no molecules have yet been approved for clinical use (Canon et al., 2019; Duffy and Crown, 2021). In these studies, the mutant RAS was targeted directly or in combination with other proteins including SOS, tyrosine kinase, SHP2, and RAF. Also, except KRASG12C mutant, the GTP-bound state of mutants has been targeted as they either lose their intrinsic or GAP-mediated GTPase activity. However, the intrinsic GTPase activity of KRASG12C is relatively higher than the other mutants which enables targeting in its GDP-bound state (Moore et al., 2020). The undruggability of RAS proteins arises from lack of deep binding pockets on the surface of the protein and also picomolar affinity of the endogenous ligands which hinders development of competitive inhibitors (Gysin et al., 2011; Ledford, 2015; Cox et al., 2014; Milroy and Ottmann, 2014). Therefore, much attention has been focused on the discovery of allosteric sites that can regulate the function of the protein (Buhrman et al., 2010; Ostrem et al., 2013; Fetics et al., 2015; Johnson et al., 2017; McCarthy et al., 2019; Khan et al., 2022). Importantly, it is well-established that the function of the protein is modulated by post-translational modifications. In particular, phosphorylation/dephosphorylation can be given as an example, which is controlled by Src-kinase and Src homology region 2 domain-containing phosphatase-2 (SHP2), respectively (Figure 1B). It has been shown that phosphorylation of the tyrosine at the 32nd position by Src-kinase attenuated RAF binding to HRAS and NRAS while elevating intrinsic GTPase activity of the proteins (Bunda et al., 2014, Figure 1B). Furthermore, recently, Kano et al. have implied that Src-kinase phosphorylated tyrosine residues at the 32nd and 64th positions of KRAS4B isoform changed conformation of Switch I and II. Consequently, this led to a decrease in intrinsic GTPase activity, thus maintaining KRAS4B in the GTP-bound state. Interestingly, phosphorylated and GTP-bound KRAS4B was shown not to bind RAF, thus leaving the protein in the dark state (Kano et al., 2019). In the same study, it was also shown that if phosphoryl groups were removed by SHP2, then GTP-bound KRAS4B could interact with RAF and initiate signaling pathways through MAPK (Kano et al., 2019). Notably, it was shown that deletion or inhibition of SHP2 could slow down tumor progression, but remaining insufficient for tumor regression (Ruess et al., 2018). Collectively, these findings suggest that mimicking dynamics invoked by phosphorylation might provide an alternative strategy for inhibiting mutant RAS/RAF interaction. In this study, we set out to investigate the impact of phosphorylation on the structure and dynamics of HRASWT by performing atomistic MD simulations. Comparison of the trajectory pertaining to the phosphorylated RAS with trajectories of GTP-bound HRASWT and HRASG12D showed that phosphorylation of Y32 increased the flexibility of RAF-RBD interface compared to the mutant and pushed Switch I, in particular Y32, out of the nucleotide-binding pocket. Considering the fact that, exposed Y32 precluded RAF binding, we searched for molecules that could evoke similar rearrangements in HRASG12D. To this end, we carried out virtual screening by using therapeutically-approved molecules deposited in DrugBank (Wishart et al., 2018; Law et al., 2014; Knox et al., 2011; Wishart et al., 2008; Wishart et al., 2006, BindingDB Gilson et al., 2016; Liu et al., 2007; Chen et al., 2001b; Chen et al., 2001a; Chen et al., 2002, DrugCentral Ursu et al., 2019; Ursu et al., 2017, and NCGC Huang et al., 2011). The impact of ligands on the structure and dynamics of HRASG12D mutant was examined using molecular dynamics simulations. We showed that cerubidine, tranilast, nilotinib, and epirubicin could induce similar dynamics and structural changes which were seen in the phosphorylated RAS protein. Moreover, we also calculated the energy required for pushing Switch I out of the nucleotide-binding pocket in the absence/presence of one of the successful ligands, namely cerubidine, using perturb-scan-pull (PSP) method Jalalypour et al., 2020 and showed that less energy was required for displacement of Switch I in the presence of the ligand. Importantly, we also tested the activity of cerubidine in preventing RAS/RAF interaction using immunoprecipitation assays and verified computational findings. Therefore, these results suggest that Y32 detachment from the nucleotide-binding pocket might be used as an alternative strategy for targeting mutant RAS proteins. Results Phosphorylation impacts the flexibility of Y32 and RAF-RBD/RAS interface residues The comparison of RMSF profiles showed remarkable differences in the fluctuation patterns of certain residues/domains among wild-type, phosphorylated, and mutant protein. We showed that phosphorylation increased the flexibility of Y32 as a result of repulsion between negatively charged phosphate and GTP and also increased the flexibility of the residues that are involved in the RAF-RBD interaction interface compared to the mutant as shown in Table 1. Since RAF-RBD plays a major role in formation of high-affinity RAS-RAF complex, increased flexibility presumably precludes RAF binding. On the other hand, phosphorylation did not impact the RAF-CRD interface, which has been shown to play an important role in anchoring RAF to the membrane (Travers et al., 2018) as revealed by NMR and mutagenesis studies (Drugan et al., 1996). It is important to note that the difference seen in the flexibility of Y32 and RAF-RBD interface residues between HRASpY32 and HRASG12D could not be observed for G60 and Q61, although the two residues could reach relatively higher RMSF values in the phosphorylated RAS (see the standard errors in Table 1). This, in turn, which might cause perturbation of the interface formed with RAF-CRD and modulate GAP-mediated GTPase activity of RAS, respectively. Table 1 The backbone RMSF values of key regions/residues pertaining to HRASWT, HRASpY32, and HRASG12D. Residue/Region-RMSF (Å)HRASWTHRASpY32HRASG12DY321.6 ± 0.21.5± 0.20.9± 0.1RAF-RBD interface residues1.2± 0.21.3± 0.20.8± 0.1RAF-CRD interface residues0.8± 0.10.9± 0.20.7± 0.03G601.0± 0.31.4± 0.51.1± 0.2Q611.2± 0.41.6± 0.41.5± 0.3 Phosphorylation pushes Switch I and Y32 out of the nucleotide-binding pocket of RAS As shown in Table 1, the flexibility of residues, which interact with RAF-CRD domain, increased upon phosphorylation compared to HRASG12D. Since these residues surround the Switch I domain, we sought to investigate whether the opening of the nucleotide-binding pocket was impacted by measuring the distance between Cα atoms of the G/D12 and P34 residues throughout the trajectories. We showed that phosphorylation pushed Switch I out of the binding pocket as the distance between G12 and P34 residues increased compared to the mutant protein (Figure 2A, Figure 2—figure supplement 1A-C). Consequently, this makes the nucleotide-binding pocket more accessible to waters, as evident from the number of waters measured within 5 Å distance of GTP: 93.60±0.20, 100.30±0.20, and 72.68±0.17 for HRAS WT, HRASpY32, and HRASG12D,respectively. Considering rearrangements occurring around the nucleotide-binding pocket and that GAP binding involves interaction with both Switch I and II, it can be said that phosphorylation triggers rearrangement around the pocket of RAS that prepares it for binding to GAP, thus, modulating the GTPase activity of the protein. Indeed, it was shown that GTPase activity of RAS increased upon phosphorylation (Bunda et al., 2014). Figure 2 with 1 supplement see all Download asset Open asset Probability distance distributions pertaining to nucleotide binding pocket and respective schematic representation of the residues used for the calculations. (a) The normalized probability density distribution of the distance measured between the Cα atom of 12th 34th residues. The standard error (SE) of the measurements for HRASpY32, HRASWT, and HRASG12D are measured as 0.01, 0.004, and 0.01 Å, respectively. (b) The normalized probability distribution of the distance between the side-chain oxygen atom of Y32 and Pγ of GTP. The SE of the measurements for HRASpY32, HRASWT, and HRASG12D are calculated as 0.02, 0.02, and 0.003 Å, respectively. (c) Cα atoms of 12th and 34th residues are shown on the crystal structure of HRASWT (PDB ID: 5P21) in vdW representation and colored with ocher and purple, respectively, whereas GTP is shown in the licorice and colored with red. (d) The orientational dynamics of Y32 in the HRASpY32 trajectory. (e) The H-bond formed between side-chain of Y32 and Pγ of GTP in HRASG12D is shown in purple. Having observed phosphorylation-induced modulation in the flexibility of Y32, we also examined the positioning of the residue by measuring the distance between the side-chain oxygen of Y32 and Pγ atom of GTP (see Figure 2—figure supplement 1D,E and F). We showed that Y32 formed a hydrogen bond with the Pγ atom of GTP in both HRASWT and HRASG12D which stabilized the residue in the vicinity of the nucleotide-binding pocket (Figure 2E). However, the hydrogen bond was not formed in HRASpY32, and Y32 was positioned far from the pocket, thus making it exposed to the environment, as evidenced by relatively longer distances measured (Figure 2B and D). In addition to the position, we also explored orientational preference of Y32 with respect to the nucleotide-binding pocket by measuring dihedral angles pertaining to backbone and side-chains of Y32, namely ϕ/ψ and χ1/χ2 angles. There was no remarkable difference in backbone dihedrals and χ1, whereas χ2 angle distribution was different among the systems studied. Specifically, Y32 displayed two peaks at –100 to –90° and 80–90° in the phosphorylated RAS, whereas it adopted 60–70° in the mutant and wild-type HRAS (Figure 3A and Figure 3—figure supplement 1). It is important to point that Y32 adopted 80° in the crystal structure of allosteric inhibitor-bound KRAS4BG12D (PDB ID:6WGN) Zhang et al., 2020, where the residue was exposed and far from the nucleotide-binding pocket as the distance measured between the side-chain of Y32 and Pγ atom of GTP was 16 Å. Figure 3 with 1 supplement see all Download asset Open asset The normalized probability density distribution plots pertaining to chi2 angle of residue Y32 along with respective snapshot. (a) The normalized probability density distribution of the measured χ2 angles of HRASWT, HRASG12D, and HRASpY32. The SE of χ2 pertaining to the phosphorylated, wild-type, and mutant systems are 0.44, 0.35, and 0.09, respectively. (b) A representative exposed state of Y32 obtained from the trajectory of the phosphorylated system. Herein, it is important to mention that exposed conformation of Y32 was not observed in the trajectories pertaining to RAF-RBD-bound HRASWT as shown in our earlier study (Ilter and Sensoy, 2019). Therefore, this finding suggests that exposure of Y32 might occlude the interaction interface formed between RAS and RAF-RBD. Global dynamics reveals a possible binding site near the nucleotide-binding pocket in HRASG12D Besides local analysis, the collective dynamic properties of the systems were also explored by calculating the principal components of their global motions. To do so, trajectories of HRASWT, HRASG12D, and HRASpY32 were projected along their first five eigenvectors, which reflect ca. 50% of the overall dynamics Petrone and Pande, 2006 (see Figure 4—figure supplement 1), and compared to each other to investigate global conformational rearrangements induced by phosphorylation and mutation. Consequently, it was shown that the dynamics of Switch I domain, in particular RAF-RBD interaction interface, could be altered in the mutant, albeit to a lesser extent, compared to both HRASWT, HRASpY32. It is also important to mention that the contribution of Y32 to the first five eigenvectors was higher in HRASpY32 (3.14 Å) than in the mutant protein (2.87 Å) as seen in the RMSF values. ON the other hand, the contribution of Switch II, which harbors both G60 and Q61, to the overall dynamics was higher in HRASWT and HRASpY32 than the mutant protein (Figure 4A). Q61 was positioned closer to the nucleotide-binding pocket in the mutant HRAS than in phosphorylated HRAS whereas G60 could sample longer distances than that was sampled by both HRASpY32 and HRASWT at their maximum probability (Figure 4C and D and Figure 4—figure supplement 2). Figure 4 with 2 supplements see all Download asset Open asset PCA analysis and Distance Probability Distributions pertaining to G60/Q61 and the nucleotide. (a) Fluctuation of Cα atoms pertaining to HRASWT, HRASG12D, and HRASpY32 along the first five eigenvectors. The RAF-CRD & -RBD interaction interfaces, as well as Switch II, are shaded in the green, pink, and black rectangles, respectively. The eigen RMSF of Y32 pertaining to the phosphorylation system is pointed out by a dark violate bead. (b) The projected trajectories of the systems studied along with the first principal component, where the thickness of the are the contribution of domain to the collective The probability density distribution of the distance between (c) the backbone of G60 and Pγ of GTP is and (d) the side-chain oxygen of Q61 and Pγ of GTP is shown. The SE of the distance between G60 and GTP pertaining to the phosphorylated, wild-type, and mutant systems are 0.01, 0.01, and Å, respectively. The SE of the distance between Q61 and GTP pertaining to the phosphorylated, wild-type, and systems are 0.02, 0.01, and 0.01, respectively. Having observed flexibility at the RAF-RBD interface in the mutant, we set out to investigate if the site can be as a possible binding pocket that can small molecules to modulate the dynamics of Switch To do so, we the trajectory of the mutant HRAS by probability density distributions of distances between the side-chain oxygen of and Pγ of GTP backbone of G60 and Pγ of which different conformational states of the nucleotide-binding pocket, to the structural studies et al., et al., 2010; et al., 2011). There were states described for as state 1, and each of which sampled distances in the range of Å, Å, and Å, respectively (Figure and Figure supplement 1). G60 could also adopt namely state 1, and which to distance range between Å, Å, and Å, respectively (Figure In of the conformation that values pertaining to 1 in each distances was up from the trajectory of HRASG12D. The possible binding pockets on the surface of mutant HRAS were and by the pocket, which relatively higher and was to be used 2). The binding pocket, which was on the conformation, was to Switch Considering the fact that this domain residues that RAF binding, as a for intrinsic GTPase activity, and the collective dynamics of the mutant the region was used as the target binding pocket in the of the Figure 5 with 1 supplement see all Download asset Open asset Probability distribution pertaining to distance measured between and G60 for the conformational state of mutant The normalized probability density distribution of the measured distance between the side-chain oxygen atom of and Pγ of and the backbone of G60 and Pγ of GTP in the mutant where the range for calculating the of each was as 1 Å. The SE of the distance between and GTP is calculated as Å, whereas that of the distance between G60 and GTP is Å. Table 2 The of possible pockets found on the surface of the conformation of HRASG12D. therapeutic molecules the RAF binding interface and pushes Y32 out of the pocket The groups of the binding site on the surface of HRASG12D were with respect to both and properties of residues DrugBank (Wishart et al., 2018; Law et al., 2014; Knox et al., 2011; Wishart et al., 2008; Wishart et al., 2006, DrugCentral Ursu et al., 2019; Ursu et al., 2017, BindingDB Gilson et al., 2016; Liu et al., 2007; Chen et al., 2001b; Chen et al., 2001a; Chen et al., 2002, and NCGC Huang et al., were searched for molecules that could at of the and have molecular than A of molecules was from the (Figure these molecules were to the binding pocket on the surface of HRASG12D and ligands were with respect to their around the nucleotide-binding pocket and which is a that is used to binding in Considering the interaction observed between Y32 and GTP in HRASG12D, we the ligands, which interaction between the nucleotide and The impact of four ligands this namely cerubidine, tranilast, nilotinib, and (Figure was tested by performing MD simulations using the 1 for respective Figure Download asset Open asset screening and molecules along with their and energy values. (a) A schematic that the virtual screening for the binding pocket on the frequently sampled conformation of HRASG12D. The and of (b) cerubidine, (c) (d) tranilast, and (e) are shown. GTP is shown in licorice and red. The trajectories were on the fluctuation of and Moreover, the distances measured between G/D12 and P34 as well as Y32 and GTP were also compared to of HRASG12D and HRASpY32. In that the of the ligands in Switch I domain, the nucleotide-binding pocket, and Y32 could be ligands, which could the flexibility of RAF-RBD and interfaces, and Switch I and Y32 from the nucleotide-binding pocket were successful in of preventing HRASG12D/RAF interaction. We showed that all the ligands, namely cerubidine, nilotinib, tranilast, and increased the flexibility of the RAF-RBD interaction interface than in HRASG12D. Moreover, the flexibility of Y32, also increased by all the ligands, except (Bunda et al., 2014; Kano et al., 1). We also examined the of the nucleotide-binding pocket and the positioning of Y32 by measuring the distances between respectively. We showed that was more to displacement of Switch I and Y32 from the nucleotide-binding pocket, whereas the impact of and epirubicin was not remarkable Figure and This, in turn, of relatively higher number of waters within the nucleotide-binding pocket in HRASG12D 1). Therefore, it is to suggest that cerubidine can the intrinsic GTPase activity of the mutant RAS by GTP to Figure with 2 supplements see all Download asset Open asset The normalized probability density distribution plots pertaining to distances of G/D12 and Y32 and chi2 angle of Y32 measured in mutant trajectories. The normalized probability density distribution of (a) the distance between Cα atoms of G/D12 and (b) the distance between the side-chain oxygen atom of Y32 and Pγ of (c) χ2 in HRASG12D, HRASpY32, and and HRASG12D. The SE of the distance pertaining to the in 0.01, 0.01, 0.02, 0.02, and Å. The SE of the same systems for distance is 0.02, 0.02, 0.02, Å, respectively. The SE of χ2 is 0.09, 0.44, and for the mutant, phosphorylated, nilotinib, and HRASG12D respectively. Also, Y32 adopted similar χ2 angle in HRASG12D to that in the phosphorylated RAS (Figure and Figure supplement 2). Considering between HRASG12D and HRASpY32, cerubidine can be to have relatively more for preventing HRASG12D/RAF interaction. Therefore, we used cerubidine in the of the study to our method reveals that displacement of Switch I and Y32 in HRASG12D is in the presence of cerubidine We set out to investigate if displacement of Switch I and Y32 is energetically favorable in the presence of the To do so, we perturb-scan-pull (PSP) method et al., which was to investigate conformational in proteins, on HRASG12D. In this and target states are described and the possible for between the and the target state is by calculating the between the The maximum is to the conformational To be with the we used the same as the distance between Cα atoms of and and the backbone of G60 and Pγ of as the which, dynamics of Switch I and II, respectively. we described and two states for Switch I and II, respectively, the obtained by of HRASG12D trajectory. for Switch I, if the measured distance between Cα atoms of and P34 is less than Å, Switch I is as in the closed state. On the other hand, the distance is 16 Å, Switch I as in the open state. The distance between and 16 Å is as the open state of Switch we also the state of the Switch II by measuring the distance between the backbone of G60 and Pγ of GTP. the distance is Å, Switch II is as in the open if in the closed state. In of