Left: Motions described by the Quasi-Mode 11 when the GDP exited on the phosphate side (blue group of pathways).

<p>These motions were highly related to those we described previously by NMA (on the right: mode 17) as putatively involved in GDP release <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002595#pcbi.1002595-Louet1" target="_blank">[13]</a>.</p

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

<div><p>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 G_α.</p> </div

Free energy profiles obtained after analysis of the WHAM.

<p>The free energies are given as a function of the distance between the GDP and the center of mass of its initial binding pocket. The dashed line represents the experimental value that was described for GDP unbinding (10 kcal.mol⁻¹).</p

Non-bonded energies of interaction between the GDP and surrounding G_α residues along representative groups of pathways (A) blue group of pathways, (B) yellow+green groups of pathways and (C) red group of pathways.

<p>On the top of the figure, key residues of G_α were colored in red or green according to their positive or negative non-bonded interactions with the ligand. On the bottom, the interaction energies between the ligand and each surrounding residues were plotted as a function of the distance covered by the GDP, every 0.02 Ångström.</p

Figure 1

<p>A) Graphical representation of the G_α subunit of the Gi heterotrimer. The Ras like and helical domains were reported in orange and black respectively. The GDP, at the interface of the two G_α sub-domains was colored according to its atom types. B) Representation of the different positions of the GDP used to extract it out from its binding pocket by the TMD approach (red, blue, yellow, green). To better appreciate the motions of the GDP along each resulting TMD trajectory (colored lines), two different views were reported on the same figure after separation of the two sub-domains of G_α.</p

Plot of the forces that were necessary to unbind the GDP out from its initial pocket along each of the TMD trajectories.

<p>Plot of the forces that were necessary to unbind the GDP out from its initial pocket along each of the TMD trajectories.</p

Solventless Mechanosynthesis of N‑Protected Amino Esters

Mechanochemical derivatizations of <i>N-</i> or <i>C-</i>protected amino acids were performed in a ball mill under solvent-free conditions. A vibrational ball mill was used for the preparation of <i>N</i>-protected α- and β-amino esters starting from the corresponding <i>N-</i>unmasked precursors via a carbamoylation reaction in the presence of di-<i>tert</i>-butyl dicarbonate (Boc₂O), benzyl chloroformate (Z-Cl) or 9-fluorenylmethoxycarbonyl chloroformate (Fmoc-Cl). A planetary ball mill proved to be more suitable for the synthesis of amino esters from <i>N</i>-protected amino acids via a <i>one-pot</i> activation/esterification reaction in the presence of various dialkyl dicarbonates or chloroformates. The spot-to-spot reactions were straightforward, leading to the final products in reduced reaction times with improved yields and simplified work-up procedures

Dissociation of Membrane-Anchored Heterotrimeric G‑Protein Induced by G_α Subunit Binding to GTP

Heterotrimeric G-proteins' activation on the intracellular side of the cell membrane is initiated by stimulation of the G-Protein Coupled Receptors (GPCRs) extra-cellular part. This two-step activation mechanism includes (1) an exchange between GDP and GTP molecules in the G_α subunit and (2) a dissociation of the whole G_αβγ complex into two membrane-anchored blocks, namely the isolated G_α and G_βγ subunits. Although X-ray data are available for both inactive G_αβγ:GDP and active G_α:GTP complexes, intermediate steps involved in the molecular mechanism of the dissociation have not yet been addressed at the molecular level. In this study, we first built a membrane-anchored intermediate G_iαβγ:GTP complex. This model was then equilibrated by molecular dynamics simulations before the Targeted Molecular Dynamics (TMD) technique was used to force the G_α subunit to evolve from its inactive (GDP-bound) to its active (GTP-bound) conformations, as described by available X-ray data. The TMD constraint was applied only to the G_α subunit so that the resulting global rearrangements acting on the whole G_αβγ:GTP heterotrimer could be analyzed. We showed how these mainly local conformational changes of G_α could initiate large domain:domain motions of the whole complex, the G_βγ behaving as an almost quasi-rigid block. This separation of the two G_α:GTP and G_βγ subunits required the loss of several interactions at the G_α:G_βγ interface that were reported. This study provided an atomistic view of the crucial intermediate step of the G-proteins activation, e.g., the dissociation, that could hardly be elucidated by the experiment

A General Approach to the Aza-Diketomorpholine Scaffold

A stereoconservative three-step synthesis to access to 1,2,4-oxadiazine-3,6-dione is presented. This underexplored platform could be considered as a constrained oxy-azapeptide or an aza-diketomorpholine, the methodology being then successfully applied to produce enantiopure aza-analogs of diketomorpholine natural products. Importantly, the first crystal structures were obtained and compared to diketomorpholine and diketopiperazine structures. Finally, a straightforward procedure concerning the coupling of this heterocyclic scaffold with various amino acids to afford original pseudodipeptide analogs was described

A General Approach to the Aza-Diketomorpholine Scaffold

A stereoconservative three-step synthesis to access to 1,2,4-oxadiazine-3,6-dione is presented. This underexplored platform could be considered as a constrained oxy-azapeptide or an aza-diketomorpholine, the methodology being then successfully applied to produce enantiopure aza-analogs of diketomorpholine natural products. Importantly, the first crystal structures were obtained and compared to diketomorpholine and diketopiperazine structures. Finally, a straightforward procedure concerning the coupling of this heterocyclic scaffold with various amino acids to afford original pseudodipeptide analogs was described