The CB1 cannabinoid receptor (CB1R) contains one of the longest N termini among class A G protein-coupled receptors. Mutagenesis studies suggest that the allosteric binding site of cannabidiol (CBD) involves residues from the N terminal domain. In order to study the allosteric binding of CBD to CB1R we modeled the whole N-terminus of this receptor using the replica exchange molecular dynamics with solute tempering (REST2) approach. Then, the obtained structures of CB1R with the N terminus were used for ligand docking. A natural cannabinoid receptor agonist, Δ9-THC, was docked to the orthosteric site and a negative allosteric modulator, CBD, to the allosteric site positioned between extracellular ends of helices TM1 and TM2. The molecular dynamics simulations were then performed for CB1R with ligands: (i) CBD together with THC, and (ii) THC-only. Analyses of the differences in the residue-residue interaction patterns between those two cases allowed us to elucidate the allosteric network responsible for the modulation of the CB1R by CBD. In addition, we identified the changes in the orthosteric binding mode of Δ9-THC, as well as the changes in its binding energy, caused by the CBD allosteric binding. We have also found that the presence of a complete N-terminal domain is essential for a stable binding of CBD in the allosteric site of CB1R as well as for the allosteric-orthosteric coupling mechanism.

Most G protein-coupled receptors that bind the hydrophobic ligands (lipid receptors and steroid receptors) belong to the most populated class A (rhodopsin-like) of these receptors. Typical examples of lipid receptors are: rhodopsin, cannabinoid (CB), sphingosine-1-phosphate (S1P) and lysophosphatidic (LPA) receptors. The hydrophobic ligands access the receptor binding site from the lipid bilayer not only because of their low solubility in water but also because of a large N-terminal domain plug preventing access to the orthosteric binding site from the extracellular milieu. In order to identify the most probable ligand exit pathway from lipid receptors CB1, S1P1 and LPA1 orthosteric binding sites we performed at least three repeats of steered molecular dynamics simulations in which ligands were pulled in various directions. For specific ligands being agonists, the supervised molecular dynamics approach was used to simulate the ligand entry events to the inactive receptor structures. For all investigated receptors the ligand entry/exit pathway goes through the gate between transmembrane helices TM1 and TM7, however, in some cases it combined with a direction toward water milieu.

Alzheimer’s disease is the most common progressive neurodegenerative disorder and is characterized by the presence of amyloid β (Aβ) plaques in the brain. The γ-secretase complex, which produces Aβ, is an intramembrane-cleaving protease consisting of four membrane proteins. In this paper we investigated the amyloidogenic fragments of amyloid precursor protein (substrates Aβ43 and Aβ45, leading to less amyloidogenic Aβ40 and more amyloidogenic Aβ42, respectively) docked to the binding site of presenilin, the catalytic subunit of γ-secretase. In total, we performed 9 μs of all-atom molecular dynamics simulations of the whole γ-secretase complex with both substrates in low (10%) and high (50%) concentrations of cholesterol in the membrane. We found that, at the high cholesterol level, the Aβ45 helix was statistically more flexible in the binding site of presenilin than Aβ43. An increase in the cholesterol concentration was also correlated with a higher flexibility of the Aβ45 helix, which suggests incompatibility between Aβ45 and the binding site of presenilin potentiated by a high cholesterol level. However, at the C-terminal part of Aβ45, the active site of presenilin was more compact in the case of a high cholesterol level, which could promote processing of this substrate. We also performed detailed mapping of the cholesterol binding sites at low and high cholesterol concentrations, which were independent of the typical cholesterol binding motifs.

Despite recent advances in research, the mechanism of Alzheimer's disease is not fully understood yet. Understanding the process of cleavage and then trimming of peptide substrates, can help selectively block γ-secretase (GS) to stop overproduction of the amyloidogenic products. Our GS-SMD server (https://gs-smd.biomodellab.eu/) allows cleaving and unfolding of all currently known GS substrates (more than 170 peptide substrates). The substrate structure is obtained by threading of the substrate sequence into the known structure of GS complex. The simulations are performed in an implicit water-membrane environment so they are performed rather quickly, 2–6 h per job, depending on the mode of calculations (part of GS complex or the whole structure). It is also possible to introduce mutations to the substrate and GS and pull any part of the substrate in any direction using the steered molecular dynamics (SMD) simulations with constant velocity. The obtained trajectories are visualized and analyzed in the interactive way. One can also compare multiple simulations using the interaction frequency analysis. GS-SMD server can be useful for revealing mechanisms of substrate unfolding and role of mutations in this process.

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