Related Experiment Videos
Guanosine triphosphatase stimulation of oncogenic Ras mutants
1Abteilung Strukturelle Biologie, Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Abstract:
Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5'-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.
Insights
Oncogenic Ras mutations impair guanosine triphosphatase (GTPase) activity, leading to cancer. A novel GTP analogue, DABP-GTP, effectively rescues this defective GTPase reaction in Ras mutants, suggesting potential for new anti-Ras drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Ras GTPase is a key regulator of cell signaling, frequently mutated in human tumors.
- Oncogenic Ras mutations, particularly at Gly12 and Gln61, lead to impaired GTPase activity and constitutive activation, driving cancer.
- Targeting the Ras GTPase reaction is a significant goal for cancer drug development.
Purpose of the Study:
- To investigate the hydrolysis of a novel guanosine 5'-triphosphate (GTP) analogue, diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP), by wild-type and oncogenic Ras mutants.
- To determine if DABP-GTP can rescue the defective GTPase activity of oncogenic Ras mutants.
- To elucidate the structural and biochemical basis for the interaction between DABP-GTP and Ras.
Main Methods:
- Biochemical assays measuring the hydrolysis of DABP-GTP by wild-type and mutant Ras proteins.
- Crystallographic studies to determine the structure of Ras bound to DABP-GTP analogues.
- Analysis of the role of specific chemical groups within DABP-GTP for its interaction with Ras.
Main Results:
- DABP-GTP is hydrolyzed by both wild-type and oncogenic Ras mutants, with significantly enhanced efficiency for common oncogenic mutants (e.g., Gly12).
- The hydrolysis reaction is independent of Gln61 and is critically dependent on an exocyclic aromatic amino group in DABP-GTP.
- Crystal structures reveal that the DABP-moiety accommodates within the Gly12/Gly13 region, bypassing a rate-limiting step in intrinsic Ras GTPase activity.
Conclusions:
- The defective GTPase switch of oncogenic Ras mutants is not irreversibly damaged and can be rescued by specific GTP analogues like DABP-GTP.
- DABP-GTP's unique chemical structure allows it to overcome the intrinsic GTPase defect in oncogenic Ras.
- These findings provide a foundation for designing novel anti-Ras drugs that target the impaired GTPase reaction.