Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy

Abstract

Time-resolved Fourier transform infrared (FTIR) spectroscopy is a powerful tool to elucidate label-free the reaction mechanisms of proteins. After assignment of the absorption bands to individual groups of the protein, the order of events during the reaction mechanism can be monitored and rate constants can be obtained. Additionally, structural information is encoded into infrared spectra and can be decoded by combining the experimental data with biomolecular simulations. We have determined recently the infrared vibrations of GTP and guanosine diphosphate (GDP) bound to Gα, a ubiquitous GTPase. These vibrations are highly sensitive for the environment of the phosphate groups and thereby for the binding mode the GTPase adopts to enable fast hydrolysis of GTP. In this study we calculated these infrared vibrations from biomolecular simulations to transfer the spectral information into a computational model that provides structural information far beyond crystal structure resolution. Conformational ensembles were generated using 15 snapshots of several 100 ns molecular-mechanics/molecular-dynamics (MM-MD) simulations, followed by quantum-mechanics/molecular-mechanics (QM/MM) minimization and normal mode analysis. In comparison with other approaches, no time-consuming QM/MM-MD simulation was necessary. We carefully benchmarked the simulation systems by deletion of single hydrogen bonds between the GTPase and GTP through several Gα point mutants. The missing hydrogen bonds lead to blue-shifts of the corresponding absorption bands. These band shifts for α-GTP (Gα-T48A), γ-GTP (Gα-R178S), and for both β-GTP/γ-GTP (Gα-K46A, Gα-D200E) were found in agreement in the experimental and the theoretical spectra. We applied our approach to open questions regarding Gα: we show that the GDP state of Gα carries a Mg, which is not found in x-ray structures. Further, the catalytic role of K46, a central residue of the P-loop, and the protonation state of the GTP are elucidated.

Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs

“…Measurement of FTIR spectra for this mutant, which were subsequently decoded by QM/MM calculations, elucidated the role of the conserved lysine. Specifically, this residue shifts negative charges from γ-GTP to β-GTP and thereby facilitates GTP hydrolysis similarly to the arginine finger, with the mutant showing similarly slowed hydrolysis kinetics (Mann et al, 2017). In conclusion, these findings reveal novel intrinsic and GAPcatalyzed hydrolysis mechanisms for G-proteins at subatomic resolution (Figure 7).…”

Microsecond resolved infrared spectroscopy on non-repetitive protein reactions by applying caged-compounds and quantum cascade laser frequency combs

“…Such a rearrangement was also observed on a slower timescale in a Gα i mutant. 27 However, further experiments including the measurements of further Gα i mutants will be necessary for a clear-cut assignment and is not within the scope of this work. The complete reaction with all the information obtained in the dual comb experiment is shown in Figure 4D.…”

The protonation states of GTP and GppNHp in Ras proteins

“…The accuracy of the determined infared vibrations is notable (1 cm -1 corresponds to a P-O bond length alteration of 0.001 nm (31, 32)) and the impact of alterations down to single hydrogen bonds was shown various times in the literature (30,33). The calculations were benchmarked against NMR spectra and infrared spectra from the literature (20,21) and were found in excellent agreement when GTP was deprotonated.…”

“…The starting structure was initially solvated in Moby following the Vedani algorithm (42) and then transferred to the GROMACS 2016 program suite (43-46) where full solvation with TIP4P waters and 154 mM NaCl in a cubic simulation cell (d=7 nm) was performed. Simulations were conducted in the OPLS/AA forcefield (47) using parameters for deprotonated GTP (27) and singly protonated GTP (33). Coulomb interactions were calculated using PME (0.9 nm) and a VDW cutoff of 1.5 nm was applied on a GPU-accelerated workstation.…”