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Published on: July 18, 2019
Structural basis of the atypical activation mechanism of KRASV14I
Asim K Bera1, Jia Lu1, Thomas E Wales2
1Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390.
Abstract:
RAS regulation and signaling are largely accomplished by direct protein-protein interactions, making RAS protein dynamics a critical determinant of RAS function. Here, we report a crystal structure of GDP-bound KRASV14I, a mutated KRAS variant associated with the developmental RASopathy disorder Noonan syndrome (NS), at 1.5-1.6 Å resolution. The structure is notable for revealing a marked extension of switch 1 away from the G-domain and nucleotide-binding site of the KRAS protein. We found that this extension is associated with a loss of the magnesium ion and a tilt in the position of the guanine base because of the additional carbon introduced by the isoleucine substitution. Hydrogen-deuterium exchange MS analysis confirmed that this conformation occurs in solution, but also disclosed a difference in kinetics when compared with KRASA146T, another RAS mutant that displays a nearly identical conformation in previously reported crystal structures. This conformational change contributed to a high rate of guanine nucleotide-exchange factor (GEF)-dependent and -independent nucleotide exchange and to an increase in affinity for SOS Ras/Rac GEF 1 (SOS1), which appears to be the major mode of activation for this RAS variant. These results highlight a mechanistic connection between KRASA146T and KRASV14I that may have implications for the regulation of these variants and for the development of therapeutic strategies to manage KRAS variant-associated disorders.
Insights
The KRAS V14I mutation causes a distinct protein structure, impacting RAS signaling and nucleotide exchange. This finding offers insights into Noonan syndrome and potential therapeutic strategies for RASopathies.
Area of Science:
- Molecular Biology
- Structural Biology
- Genetics
Background:
- RAS proteins are key regulators of cellular signaling, with their function critically dependent on dynamic protein interactions.
- Mutations in RAS genes are linked to various developmental disorders, including RASopathies like Noonan syndrome.
Purpose of the Study:
- To determine the crystal structure of GDP-bound KRAS V14I, a variant associated with Noonan syndrome.
- To elucidate the structural and functional consequences of the V14I mutation on KRAS protein dynamics and signaling.
Main Methods:
- X-ray crystallography to determine the high-resolution structure of KRAS V14I.
- Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to analyze protein conformation in solution.
- Biochemical assays to assess nucleotide exchange rates and GEF binding affinity.
Main Results:
- The KRAS V14I structure revealed an extended switch 1 region, loss of magnesium ion, and altered guanine base positioning.
- HDX-MS confirmed this conformation in solution and showed distinct kinetics compared to KRAS A146T.
- The mutation led to increased nucleotide exchange rates and enhanced affinity for SOS Ras/Rac GEF 1 (SOS1).
Conclusions:
- The V14I mutation induces a specific conformational state in KRAS that promotes nucleotide exchange and SOS1 interaction, representing a major activation pathway.
- This study reveals a mechanistic link between KRAS V14I and KRAS A146T, with implications for understanding RAS variant regulation.
- Findings may inform the development of targeted therapies for RAS variant-associated disorders.
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