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In Silico Screening of NRas Protein Oncogenic Mutations: New Structural and Physico-Chemical Insights into the Catalytic Activity

  • Tichauer, Ruth
Publication Date
Feb 01, 2019
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By controlling cellular signal transduction pathways, Ras proteins play a key role in cell differentiation, proliferation and survival. In the GTP-bound conformation, they interact with downstream effector molecules ensuring multiple tasks within the cell. The hydrolysis reaction of GTP into GDP that is catalyzed in their center, is accompanied by a conformational change, leading to an inactive state. Specific Ras mutations cause a hydrolysis rate drop that leaves Ras in a GTP-bound, continuously active, state. Now, these mutated hyperactive forms of Ras have been associated with abnormal cell proliferation, emblematic of cancerous tissues dissemination. Therefore, the elucidation of Ras catalyzing mechanisms for accelerating GTP hydrolysis constitutes a major step in the development of cancer targeted therapies. In this sense, we attempted to understand such mechanisms and propose a new strategy to restore, within oncogenic mutants, a hydrolysis rate close to that of the wild-type protein. This study is focused on NRas isoform that is mutated in 25% of malignant melanomas. In order to get insight at the atomic level on Ras catalyzing properties, molecular dynamics simulations were carried out, describing wild-type and mutant NRas active site at different levels of theory (Molecular Mechanics, Semi-empirical and Density Functional Theory). These calculations first show that water molecules positioning in the active site is crucial for an efficient catalysis of the reaction. Indeed, the precise solvent distribution observed within the wild-type is lost within the mutated forms of NRas considered here. This difference, as well as the active site structural modifications induced by Gln 61 substitutions, have a direct impact on GTP electronic density that is accommodated to a GDP-like state within the wild-type protein only. To end, an alternative reaction pathway of the enzymatic hydrolysis of GTP is proposed. Besides, these calculations were coupled to biomechanical characterizations, employing the Static Modes approach. They lead to the identification of residues of interest that have a mechanical influence on Ras GTPase function. Subsequently, mutations were introduced on these sites to identify an additional mutation that would annihilate the effects of the primary oncogenic mutations. The characterization of the doubly mutated proteins shows that one of them restores water distribution so that it is similar to that observed within wild-type NRas. Moreover, this function recovery engineered protein has been validated in cellulo by an experimental team. This result should make it possible to propose new drug molecules that mimick the effects of the second mutation and thereby restore GTP hydrolysis within oncogenic NRas.

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