Abstract
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.
Highlights
In the intracellular compartment, activation of membraneanchored heterotrimeric G-proteins involves an exchange between GDP and GTP molecules in the Ga subunit
Many X-ray structures are available in the Protein Data Bank that describe either G-proteins [5,6], G-Protein Coupled Receptors (GPCRs) [7,8], or more recently, their putative interactions [9], the possible mechanism of GDP release still remains unknown at the molecular level
In this study we were interested in the unbinding process of GDP from the Giabc complex by using extensive molecular dynamics (MD) simulations and reconstruction of free energy profiles along the different putative exit pathways, for a total simulation time overcoming 500 ns (Figure 2)
Summary
Activation of membraneanchored heterotrimeric G-proteins involves an exchange between GDP and GTP molecules in the Ga subunit. GDP release out of Ga is ‘‘catalyzed’’ by the direct interaction of the whole heterotrimer with an activated G-protein Coupled Receptor (GPCR) and appears as the rate limiting step [2] This interaction mainly involves the Cterminal helix of Ga as shown by biochemical and structural data [3,4]. The X-Ray structure of the complex between Gsabc and the beta-2 adrenergic receptor brought some elements on the GDP-free state of an heterotrimeric G-protein in complex with a GPCR [9]. In this study we were interested in the unbinding process of GDP from the Giabc complex by using extensive molecular dynamics (MD) simulations and reconstruction of free energy profiles along the different putative exit pathways, for a total simulation time overcoming 500 ns (Figure 2). The forced extraction of GDP on this side promoted large amplitude motions of the protein that were in close agreement with those we described previously as putatively involved in GDP release [13]
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