The protonation states of GTP and GppNHp in Ras proteins

Abstract

The small GTPase Ras transmits signals in a variety of cellular signaling pathways, most prominently in cell proliferation. GTP hydrolysis in the active center of Ras acts as a prototype for many GTPases and is the key to the understanding of several diseases, including cancer. Therefore, Ras has been the focus of intense research over the last decades. A recent neutron diffraction crystal structure of Ras indicated a protonated γ-guanylyl imidodiphosphate (γ-GppNHp) group, which has put the protonation state of GTP in question. A possible protonation of GTP was not considered in previously published mechanistic studies. To determine the detailed prehydrolysis state of Ras, we calculated infrared and NMR spectra from quantum mechanics/molecular mechanics (QM/MM) simulations and compared them with those from previous studies. Furthermore, we measured infrared spectra of GTP and several GTP analogs bound to lipidated Ras on a membrane system under near-native conditions. Our findings unify results from previous studies and indicate a structural model confirming the hypothesis that γ-GTP is fully deprotonated in the prehydrolysis state of Ras.

Keywords: Fourier transform IR, FTIR; GTP hydrolysis; GTPase; NMR; QM/MM simulation; Ras protein; molecular dynamics; nuclear magnetic resonance; quantum chemistry.

Figure 1.

Neutron diffraction crystal structure of Ras with protonated γ-GppNHp. Observed deuterons of the triphosphate groups are depicted as white spheres (PDB ID: 4RSG). Tyr-32 of a neighboring symmetry cell is bound to γ-GppNHp and is depicted as blue sticks. The Mg²⁺ atom is depicted as a green sphere. Labels of α-,β-, and γ-oxygens were retained hereafter. Protonation at γ2 (asterisk) corresponds to experimental observation of γ-protonation.

Figure 2.

Infrared spectra of GTP, GppNHp, GppCH₂p, and GTPγS bound to Ras on a membrane system using ATR-FTIR measurements. A, lipidated N-Ras was immobilized on a POPC membrane in an ATR-FTIR setup. By buffer exchange in the flow-through system nucleotide exchange can be performed. B, comparison to previous transmission FTIR experiments with H-Ras (inverted, scaled by 0.01) show identical band positions. C, nucleotide exchange to GTP, GppNHp, GppCH₂p, and GTPγS in ATR-FTIR was performed with N-Ras·GDP background.

Figure 3.

Comparison: Ras·GppNHp on ATR-FTIR and transmission-FTIR. To verify the ATR-FTIR experiments, we performed transmission FTIR experiments with photocaged GppNHp. Bands of α-GppNHp and β-GppNHp were identical, but γ-GppNHp showed two asymmetrical stretching vibrations instead of only one observed vibration in the ATR-FTIR measurements. Isotopic labeling assigned the negative band at 1137 cm⁻¹ to β-GDP. Therefore, the γ-GppNHp band at 1128 cm⁻¹ was superimposed by β-GDP in the ATR-FTIR measurements and reduced to a shoulder. Hence γ-GppNHp owns two asymmetrical stretching vibrations at 1128 cm⁻¹ and 1114 cm⁻¹.

Figure 4.

NMR and infrared spectra for deprotonated and protonated GTP. A and D, geometries of (A) deprotonated and (D) γ-protonated GTP. Shown are the crystal structures of (A) Ras·GTP (1QRA.pdb) and (D) Ras·GppNHp (4RSG.pdb) in color and the dynamics of the substrate in the QM/MM optimized snapshots (gray sticks). The γ-GTP proton sampled the crystal structure (black asterisk), an O–Pγ–O–H torsion angle of 0° (orange asterisk) and 180° (cyan asterisk). B, calculated ³¹P NMR spectra match the experimental spectra for deprotonated GTP, with Pβ being most shielded, followed by Pα and Pγ, respectively. E, this was not the case for protonated GTP. C and F, the same applies for infrared spectra of deprotonated (C) and protonated (F) GTP. Therefore, GTP was most likely deprotonated in the experiments. Error bars depict the mean ± S.E. from each 15 independent calculations.

Figure 5.

NMR and IR spectra for GTP protonation on all possible sites. We calculated NMR and IR spectra for fully deprotonated phosphate groups and every possible protonation (γ1/γ2/γ3, β1/β2/, α1/α2; atom names match those in Fig. 1) of GTP bound to Ras (M06/6–31G*) from the crystal structure 1QRA.pdb. Results for deprotonated GTP and γ2 protonated GTP agree well to the values calculated from 15 QM/MM snapshots. Most significant deviations from experimental values are indicated by red circles. NMR spectra show significant deviations for γ protonation and α protonation. IR spectra show significant deviations for every kind of protonation, indicating fully deprotonated triphosphate was present in the experiments.

Figure 6.

Calculation of GTP, GppNHp, and GppCH₂p protonation. A and B, we calculated protonated and deprotonated NMR (A) and IR spectra (B) of Ras·GTP, Ras·GppNHp, and Ras·GppCH₂p. The depicted γ protonations correspond to γ2 in Fig. 1 similarly to Ref. . Protonation always caused deviation from the experimental values. Protonated Ras·GppCH₂p always deprotonated during the calculations (asterisks).