Guanosine triphosphatase stimulation of oncogenic Ras mutants

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.

Figure 1

Chemical structure of the GTP-phosphonoamidate derivatives. MABP, monoaminobenzophenone; PMA, phenylene-monoamine.

Figure 2

HPLC and fluorescence measurements of the hydrolysis reaction of Ras-proteins loaded with either GTP or GTP-analogues. (a) Single turnover hydrolysis of 150 μM wild-type Ras (closed symbols) and A61Ras (open symbols) with 140 μM of either GTP (triangles) or DABP-GTP (circles) was measured in standard buffer at 30°C. Aliquots of the reaction mixture at the indicated time points were analyzed by HPLC as described in Materials and Methods. (b) HPLC analysis of DABP-GTPase reaction products by wild-type Ras after 0, 1, 5, and 30 min. Elution times of standards GDP (3.7 min), DABP-GTP (5.7 min), and DABP-P_i (7.3 min) are indicated. (c) Real time tryptophane fluorescence kinetics of the hydrolysis of GTP and various GTP-derivatives illustrated in Fig. 1 were followed by rapid-mixing of 1.1 μM nucleotide-free W32Ras with 1 μM of the respective GTP-analogues in standard buffer at 30°C by using stopped-flow apparatus. For calculation of the rate constants, the data were fitted to a single exponential.

Figure 3

HPLC and fluorescence measurements of the hydrolysis reaction of GTP and DABP-GTP by oncogenic Ras. (a) GTPase (full symbols) and DABP-GTPase (open symbols) of V12Ras (triangles), R12Ras (circles), and A13Ras (squares) were analyzed by HPLC as before. (b) Time course of tryptophane fluorescence of the DABP-GTPase reaction of oncogenic Ras mutants (as indicated) in W32-background was measured as before. As an internal control, the GTPase of W32V12Ras is shown. The data were analyzed by single exponentials.

Figure 4

Three-dimensional view of the active site of the Ras(G12P)⋅DABP-GppNHp complex showing the P-loop containing the G12P mutation, the visible part of switch I, part of switch II, the Mg²⁺ ion, and several water molecules, as well as DABP-GppNHp.

Figure 5

Solvent isotope effect of the GTPase and DABP-GTPase reaction. W32Ras was incubated with DABP-GTP in either H₂O or D₂O as indicated. The reaction was monitored by the fluorescence increase as before. The data were analyzed by single exponentials.