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The 2-D plot showing the projections of wild-type (green) and mutant (red) trajectories, using multivariate PCA approach. A combination of dihedral, interactions and energy were used as features, as labelled on the X and Y axis. https://doi.org/10.1371/journal.pone.0234836.g008
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The Ras family of proteins is known to play an important role in cellular signal transduction. The oncoprotein Ras is also found to be mutated in ~90% of the pancreatic cancers, of which G12V, G13V, A59G and Q61L are the known hot-spot mutants. These ubiquitous proteins fall in the family of G-proteins, and hence switches between active GTP bound a...
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... MD simulation based studies of wild-type and hot-spot mutant A59G HRas trajectories as listed: F and ψ dihedrals of Gln61 and Tyr64 residues, crucial interactions between Tyr64-Tyr32, Tyr64-Thr35 and Tyr64-Gln61 and the total energy component as calculated from the MMGBSA energy analysis (S1 File). Fig 8 shows the projections of these features in the two-dimensional matrix, where the wild-type system is represented in green clusters and the mutant system in red clusters. The plot shows that the ψ dihedral of Gln61 and Tyr64 along with the distances and energy component were able to distinguish the wildtype and mutant ensembles. ...
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Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anti-cancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activity or mediates its downstream signaling. To this end, we used a...
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... Owing to the fast exchange of GTP the crystal structures have been solved in presence of its analogues namely GCP, GNP and GSP [3]. The interaction between SOS1 and KRAS plays an important role in transmitting allosteric signals and is also seen from various experimental studies on KRAS, SOS1, KRAS-SOS1 where researchers have tried to identify interaction networks between these proteins [16], [24], [27], [28]. Besides experimental studies, various in silico studies have also been performed to understand the SOS1 mediated KRAS activation and network interactions responsible in the activation process [28][29][30][31][32]. Researchers had studied both KRAS and SOS1 individually and their intrinsic interaction network, but details of SOS1 mediated KRAS activation mechanism are still not clear [33], [34]. ...
... The distance and energetics analysis shows that the presence of GDP/GTP bound KRAS REM may have led to conformational changes that differentially regulates the KRAS CDC25 . To and G60 with GDP are known to be greater as compared to their distances with GTP, suggesting state 1 and state 2, respectively [27], [55]. In order to observe the variation in these distances, they were calculated for all the nine systems which have been shown in Figure 5A-C. ...
KRAS activation is known to be modulated by a guanine nucleotide exchange factor (GEF), namely, Son of Sevenless1 (SOS1). SOS1 facilitates the exchange of GDP to GTP thereby leading to activation of KRAS. The binding of GDP/GTP to KRAS at the REM/allosteric site of SOS1 regulates the activation of KRAS at CDC25/catalytic site by facilitating its exchange. Different aspects of the allosteric activation of KRAS through SOS1 are still being explored. To understand the SOS1 mediated activation of KRAS, molecular dynamics simulations for a total of nine SOS1 complexes (KRAS-SOS1-KRAS) were performed. These nine systems comprised different combinations of KRAS-bound nucleotides (GTP/GDP) at REM and CDC25 sites of SOS1. Various conformational and thermodynamic parameters were analyzed for these simulation systems. MMPBSA free energy analysis revealed that binding at CDC25 site of SOS1 was significantly low for GDP-bound KRAS as compared to that of GTP-bound KRAS. It was observed that presence of either GDP/GTP bound KRAS at the REM site of SOS1 affected the activation related changes in the KRAS present at CDC25 site. The conformational changes at the catalytic site of SOS1 resulting from GDP/GTP-bound KRAS at the allosteric changes may hint at KRAS activation through different pathways (slow/fast/rare). The allosteric effect on activation of KRAS at CDC25 site may be due to conformations adopted by switch-I, switch-II, beta2 regions of KRAS at REM site. The effect of structural rearrangements occurring at allosteric KRAS may have led to increased interactions between SOS1 and KRAS at both the sites. The SOS1 residues involved in these important interactions with KRAS at the REM site were R694, S732 and K735. Whereas the ones interacting with KRAS at CDC25 site were S807, W809 and K814. This may suggest the crucial role of these residues in guiding the allosteric activation of KRAS at CDC25 site. The conformational shifts observed in the switch-I, switch-II and alpha3 regions of KRAS at CDC25 site may be attributed to be a part of allosteric activation. The binding affinities, interacting residues and conformational dynamics may provide an insight into development of inhibitors targeting the SOS1 mediated KRAS activation.
... Experimental kinetic measurements are nevertheless widely available for WT and mutant Ras proteins, 12,22,38 pointing to the loss of catalytic activity due to the impaired rate of hydrolysis. Computational studies elaborated on the changes in the reactant state (RS, Figure 3A) Ras.GTP complex structures upon Gly12, and Gln61 mutations, [39][40][41][42][43][44][45] including in-depth analysis of the changes in atomic charges and the polarization of the active site before the reaction. 46 However, calculations to evaluate the influence of the important oncogenic changes on the reaction mechanism are missing. ...
Ras-positive cancer constitutes a major challenge for medical treatment. Hot spot residues Gly12, Gly13 and Gln61 constitute the majority of oncogenic mutations which are associated with detrimental clinical prognosis. Here we pre-sent a two-step mechanism of GTP hydrolysis of the wild type Ras.GAP complex using QM/MM free energy calculations with the finite-temperature string method. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Bornsted base. We obtained reaction profiles for key oncogenic Ras mutants G12D and G12C, reproducing the experimentally observed loss of catalytic activity, and validating our reaction mechanism. Using the optimized reaction path,we devised a fast and accurate simplified QM/MM reaction path optimization procedure, to design GAP mutants that activate G12D Ras. We identified 10 GAP residues that we mutated to any other possible amino acids (except for Gly), and the activation barrier was determined for 180 single mutants. Our simplified protocol gave excellent accuracy with the full QM/MM optimized paths for all but 1 outlier on top selected GAP mutants. To further enable ultra-fast screening, we built a machine learning framework to perform a fast prediction of the barrier heights, which was tested both on the single mutation data as well as on top predicted double mutations. Our approach enables a fast and accurate screening at the level of DFT-based QM/MM reaction path optimizations to design protein sequences that help restore catalytic activity of oncogenic Ras.
... Experimental kinetic measurements are nevertheless widely available for WT and mutant Ras proteins, 12,22,36 pointing to the loss of catalytic activity due to the impaired rate of hydrolysis. Computational studies elaborated on the changes in the reactant state (RS, Figure 3A) Ras.GTP complex structures upon Gly12, and Gln61 mutations, [37][38][39][40][41][42][43] including in-depth analysis of the changes in atomic charges and the polarization of the active site before the reaction. 44 However, calculations to evaluate the influence of the important oncogenic changes on the reaction mechanism are missing. ...
Ras-positive cancer constitutes a major challenge for medical treatment. Hot spot residues Gly12, Gly13 and Gln61 constitute the majority of oncogenic mutations which are associated with detrimental clinical prognosis. Here we present a two-step mechanism of GTP hydrolysis of the wild type Ras.GAP complex using QM/MM free energy calculations with the finite-temperature string method. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Brønsted base. We obtained reaction profiles for key oncogenic Ras mutants G12D and G12C, reproducing the experimentally observed loss of catalytic activity, and validating our reaction mechanism.
Markov state models (MSM) are a popular statistical method for analyzing the conformational dynamics of proteins including protein folding. With all statistical and machine learning (ML) models, choices must be made about the modeling pipeline that cannot be directly learned from the data. These choices, or hyperparameters, are often evaluated by expert judgment or, in the case of MSMs, by maximizing variational scores such as the VAMP-2 score. Modern ML and statistical pipelines often use automatic hyperparameter selection techniques ranging from the simple, choosing the best score from a random selection of hyperparameters, to the complex, optimization via, e.g., Bayesian optimization. In this work, we ask whether it is possible to automatically select MSM models this way by estimating and analyzing over 16,000,000 observations from over 280,000 estimated MSMs. We find that differences in hyperparameters can change the physical interpretation of the optimization objective, making automatic selection difficult. In addition, we find that enforcing conditions of equilibrium in the VAMP scores can result in inconsistent model selection. However, other parameters that specify the VAMP-2 score (lag time and number of relaxation processes scored) have only a negligible influence on model selection. We suggest that model observables and variational scores should be only a guide to model selection and that a full investigation of the MSM properties should be undertaken when selecting hyperparameters.
Ras GTPases play a crucial role in cell signaling pathways. Mutations of the Ras gene occur in about one third of cancerous cell lines and are often associated with detrimental clinical prognosis. Hot spot residues Gly12, Gly13, and Gln61 cover 97% of oncogenic mutations, which impair the enzymatic activity in Ras. Using QM/MM free energy calculations, we present a two-step mechanism for the GTP hydrolysis catalyzed by the wild-type Ras.GAP complex. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Brønsted base. We also determined the reaction profiles for key oncogenic Ras mutants G12D and G12C using QM/MM minimizations, matching the experimentally observed loss of catalytic activity, thereby validating our reaction mechanism. Using the optimized reaction paths, we devised a fast and accurate procedure to design GAP mutants that activate G12D Ras. We replaced GAP residues near the active site and determined the activation barrier for 190 single mutants. We furthermore built a machine learning for ultrafast screening, by fast prediction of the barrier heights, tested both on the single and double mutations. This work demonstrates that fast and accurate screening can be accomplished via QM/MM reaction path optimizations to design protein sequences with increased catalytic activity. Several GAP mutations are predicted to re-enable catalysis in oncogenic G12D, offering a promising avenue to overcome aberrant Ras-driven signal transduction by activating enzymatic activity instead of inhibition. The outlined computational screening protocol is readily applicable for designing ligands and cofactors analogously.
Costello syndrome is a clinically recognizable, severe neurodevelopmental disorder caused by heterozygous activating variants in HRAS. The vast majority of affected patients share recurring variants affecting HRAS codons 12 and 13 and a relatively uniform phenotype. Here, we report the unique and attenuated phenotype of six individuals of an extended family affected by the HRAS variant c.176C>T p.(Ala59Gly), which, to our knowledge, has never been reported as a germline variant in patients so far. HRAS Alanine 59 has been previously functionally investigated as an oncogenic hotspot and the p.Ala59Gly substitution was shown to impair intrinsic GTP hydrolysis. All six individuals we report share a phenotype of ectodermal anomalies and mild features suggestive of a RASopathy, reminiscent of patients with Noonan syndrome-like disorder with loose anagen hair. All six are of normal intelligence, none have a history of failure to thrive or malignancy, and they have no known cardiac or neurologic pathologies. Our report adds to the previous reports of patients with rare variants affecting amino acids located in the SWITCH II/G3 region of HRAS and suggests a consistent, attenuated phenotype distinct from classical Costello syndrome. We propose the definition of a new distinct HRAS-related RASopathy for patients carrying HRAS variants affecting codons 58, 59, 60.
