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Mutations in the GTPase enzyme K-Ras, specifically at codon G12, remain the most common genetic alterations in human cancers. The mechanisms governing activation of downstream signaling pathways and how they relate back to the identity of the mutation have yet to be completely defined. Here we use native mass spectrometry (MS) combined with ultraviolet photodissociation (UVPD) to investigate the impact of three G12X mutations (G12C, G12V, G12S) on the homodimerization of K-Ras as well as heterodimerization with a downstream effector protein, Raf. Electrospray ionization (ESI) was used to transfer complexes of WT or G12X K-Ras bound to guanosine 5′-diphosphate (GDP) or GppNHp (non-hydrolyzable analogue of GTP) into the gas phase. Relative abundances of homo- or hetero-dimer complexes were estimated from ESI-MS spectra. K-Ras + Raf heterocomplexes were activated with UVPD to probe structural changes responsible for observed differences in the amount of heterocomplex formed for each variant. Holo (ligand-bound) fragment ions resulting from photodissociation suggest the G12X mutants bind Raf along the expected effector binding region (β-interface) but may interact with Raf via an alternative α-interface as well. Variations in backbone cleavage efficiencies during UV photoactivation of each variant were used to relate mutation identity to structural changes that might impact downstream signaling. Specifically, oncogenic upregulation for hydrogen-bonding amino acid substitutions (G12C, G12S) is achieved by stabilizing β-interface interactions with Raf, while a bulkier, hydrophobic G12V substitution leads to destabilization of this interface and instead increases the proximity of residues along the α-helical bundles. This study deciphers new pieces of the complex puzzle of how different K-Ras mutations exert influence in downstream signaling.
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Allosteric site variants affect GTP hydrolysis on Ras
Abstract RAS GTPases are proto‐oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP‐bound and GTP‐bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF‐RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP‐bound state of RAS.
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- Award ID(s):
- 2121426
- PAR ID:
- 10464029
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 32
- Issue:
- 10
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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