Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

Abstract

Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P(i) by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10(5) compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10(5) compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with time-resolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ- relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α- relative to the β- and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.

Fig. 1.

Simulation. The prepared X-ray structures were relaxed by MM simulations in a water box (grey) with physiological NaCl (green and yellow spheres). The Ras•GAP (light blue and green, respectively) complex simulation system is shown. Structures equilibrated by MM were used as starting structures in QM/MM simulations. To examine charge distribution and geometry below the resolution of X-ray analysis, crucial parts of the hydrolysis reaction were treated quantum mechanically. The enlargement shows all atoms treated quantum mechanically. The rest of the protein and the solvent were treated by MM. In the QM/MM equilibration runs, the side chains of Ser17 and Thr35 and the two coordinating waters were not treated by QM. In the QM/MM production runs, the ribose was not treated by QM.

Fig. 2.

Comparison of theoretical and experimental difference spectra of Ras•GTP•Mg²⁺ and Ras•GTP•Mg²⁺. To compare directly the bandwidth and relative positions between the GTP and GDP states, we imposed a 20-cm^-1 shift to higher wavenumbers on the simulated vibrational modes below 1,150 cm^-1. The vibrational modes were calculated without a scaling factor. The intensities are normalized. The calculated bandwidth is the standard deviation of the frequencies caused by the inhomogeneous broadening taken from six snapshots for each state. The overlay reveals good agreement between theory and experiment for bandwidth and intensity. The grey-hatched band in the experimental spectrum is evoked by the P_i, which is not included in the simulation and therefore cannot be observed in the calculated spectra. Detailed frequencies are given in Table S1.

Fig. 3.

Changes in the triphosphate conformation that accelerate hydrolysis by destabilizing the reactant state. Shown on the left is the triphosphate with the perspective along the axis through the γ- and the β-phosphate. Shown on the right is the triphosphate with the perspective along the axis through the α- and the β-phosphate. The protein environment of Ras changes the conformation of GTP•Mg²⁺ from a staggered position of the nonbridging oxygen atoms of β- and γ-phosphate in water (A) to an eclipsed one in Ras (B). In the Ras•GAP complex (C), the intruding arginine finger of GAP further leads to an eclipsed position of the β- and α-phosphates.

Fig. 4.

Changes in charge distribution and structure of the triphosphate that accelerate hydrolysis. The γ-phosphate becomes more positive, facilitating the positioning of the negatively charged oxygen of the attacking water, and two of the nonbridging oxygen atoms of the γ-phosphate remain negative, presenting possible targets for proton transfer. The bond between P₃ and O₃₂ that is cleaved during hydrolysis is lengthened, and the γ-phosphate group becomes more planar. These effects already occur in Ras and are amplified by complex formation with GAP. All partial charges are given in the unit of the electron charge, e₀.

Fig. 5.

Changes in geometry and charge responsible for catalyzing the increase in GTP hydrolysis by 10 orders of magnitude. The Ras protein forces GTP into a conformation with a conformational energy 154 kJ/mol higher than that of GTP in water. This is effected by a change from the staggered to the eclipsed positioning of the nonbridging oxygen atoms of the γ- and β-phosphates (induced by the change from an α-, β-, and γ-phosphate–coordinated Mg²⁺ in water to a β- and γ-phosphate–coordinated one in Ras), as well as an elongation of the bond between P₃ and O₃₂ that is cleaved during hydrolysis, and the γ-phosphate group’s becoming more planar. These effects are amplified by the intruding arginine finger of GAP, and the nonbridging oxygen atoms of the α- and β-phosphates are also rotated into an eclipsed position. These lead to a further 28 kJ/mol increase in the conformational energy of GTP bound to Ras•GAP compared to GTP bound to Ras. This is similar to the experimentally determined 26 kJ/mol difference in the free energies of activation for hydrolysis in Ras (30 min) and in Ras•GAP (50 ms). This indicates acceleration by a factor of 10⁵ from Ras to Ras•GAP and by 10¹⁰ from water to Ras•GAP.