GDP Release Preferentially Occurs on the Phosphate Side in Heterotrimeric G-proteins

After extra-cellular stimulation of G-Protein Coupled Receptors (GPCRs), GDP/GTP exchange appears as the key, rate limiting step of the intracellular activation cycle of heterotrimeric G-proteins. Despite the availability of a large number of X-ray structures, the mechanism of GDP release out of heterotrimeric G-proteins still remains unknown at the molecular level. Starting from the available X-ray structure, extensive unconstrained/constrained molecular dynamics simulations were performed on the complete membrane-anchored Gi heterotrimer complexed to GDP, for a total simulation time overcoming 500 ns. By combining Targeted Molecular Dynamics (TMD) and free energy profiles reconstruction by umbrella sampling, our data suggest that the release of GDP was much more favored on its phosphate side. Interestingly, upon the forced extraction of GDP on this side, the whole protein encountered large, collective motions in perfect agreement with those we described previously including a domain to domain motion between the two ras-like and helical sub-domains of Gα. Despite the availability of many structural and biochemical data, the activation of G-proteins remains to be understood at the molecular level. We used a computation tool to decipher the first limiting step of this activation: GDP release. Combining different methods of analysis, we propose that the GDP exit occurs on its phosphate side. This study helped to rationalize some experimental observations from the literature and opens many perspectives concerning the study of G-proteins activation and their putative inhibition.