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Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein
Published on: January 16, 2020
Identification of functional substates of KRas during GTP hydrolysis with enhanced sampling simulations
Juan Zeng1, Jian Chen2, Fei Xia2
1School of Biomedical Engineering, Guangdong Medical University, Dongguan 523808, China.
Abstract:
As the hub of major signaling pathways, Ras proteins are implicated in 19% of tumor-caused cancers due to perturbations in their conformational and/or catalytic properties. Despite numerous studies, the functions of the conformational substates for the most important isoform, KRas, remain elusive. In this work, we perform an extensive simulation analysis on the conformational landscape of KRas in its various chemical states during the GTP hydrolysis cycle: the reactant state KRasGTP·Mg2+, the intermediate state KRasGDP·Pi·Mg2+ and the product state KRasGDP·Mg2+. The results from enhanced sampling simulations reveal that State 1 of KRasGTP·Mg2+ has multiple stable substates in solution, one of which might account for interacting with GEFs. State 2 of KRasGTP·Mg2+ features two substates "Tyr32in" and "Tyr32out", which are poised to interact with effectors and GAPs, respectively. For the intermediate state KRasGDP·Pi·Mg2+, Gln61 and Pi are found to assume a broad set of conformations, which might account for the weak oncogenic effect of Gln61 mutations in KRas in contrast to the situation in HRas and NRas. Finally, the product state KRasGDP·Mg2+ has more than two stable substates in solution, pointing to a conformation-selection mechanism for complexation with GEFs. Based on these results, some specific inhibition strategies for targeting the binding sites of the high-energy substates of KRas during GTP hydrolysis are discussed.
Insights
Ras proteins, crucial in cancer, have elusive KRas functions. Simulations reveal KRas substates during GTP hydrolysis, offering insights into GEF, effector, and GAP interactions and potential cancer drug targets.
Area of Science:
- Biochemistry
- Molecular Biology
- Computational Biology
Background:
- Ras proteins are central to cell signaling and implicated in 19% of cancers.
- Understanding KRas conformational substates is key, as their functions remain unclear.
- KRas mutations contribute to tumorigenesis, highlighting the need for detailed functional analysis.
Purpose of the Study:
- To extensively simulate and analyze the conformational landscape of KRas.
- To investigate KRas substates in its GTP-hydrolysis cycle states: reactant (KRasGTP·Mg2+), intermediate (KRasGDP·Pi·Mg2+), and product (KRasGDP·Mg2+).
- To elucidate the functional roles of KRas conformational substates in protein interactions.
Main Methods:
- Enhanced sampling simulations were employed to explore the conformational landscape of KRas.
- Analysis focused on the distinct chemical states of KRas during GTP hydrolysis.
- Computational modeling was used to identify stable substates and their potential interaction interfaces.
Main Results:
- KRasGTP·Mg2+ (State 1) exhibits multiple substates, including one for GEF interaction.
- KRasGTP·Mg2+ (State 2) has "Tyr32in" and "Tyr32out" substates for effector and GAP binding, respectively.
- KRasGDP·Pi·Mg2+ shows conformational flexibility in Gln61 and Pi, potentially explaining weaker oncogenic effects of Gln61 mutations.
- KRasGDP·Mg2+ possesses multiple substates, suggesting a conformation-selection mechanism for GEF binding.
Conclusions:
- KRas conformational substates play distinct roles in regulating its interactions with GEFs, effectors, and GAPs.
- The conformational flexibility of KRas during GTP hydrolysis is critical for its function.
- Targeting high-energy KRas substates during GTP hydrolysis presents a potential cancer inhibition strategy.
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