Theoretical IR Spectroscopy Based on QM/MM Calculations Provides Changes in Charge Distribution, Bond Lengths, and Bond Angles of the GTP Ligand Induced by the Ras-Protein

The GTPase Ras p21 is a crucial switch in cellular signal transduction. Fourier transform infrared (FTIR) spectra of the substrate guanosine triphosphate (GTP) show remarkable changes when it binds to the enzyme. The reduced band widths indicate that the flexible GTP molecule is guided by the protein into a preferred conformation. The delocalized phosphate vibrations of unbound GTP become localized. The frequency shifts show an electron movement toward β-phosphate, which probably contributes to catalysis by reducing the free activation energy. To quantify these qualitative observations we performed QM/MM molecular dynamics simulations of Ras·GTP and GTP in water. The triphosphate part of GTP was treated quantum mechanically using density functional theory (DFT). Vibrational spectra were calculated in harmonic approximation with an average deviation of 3% from the experimental frequencies. This provides a high confidence in the computational results as vibrational spectra are highly sensitive to conformation and charge distribution. As compared to GTP in water, Ras-bound GTP shows a shift of negative charge of ∼0.2 e toward the β-phosphate from γ-phosphate and from α-phosphate due to the positive charge of the magnesium ion, to a lesser extent of Lys-16, and surprisingly without any effect of the P-loop backbone. Magnesium and Gly-13 twist and bend the γ-O-β bonds such that the crucial bond is stretched before cleaving.