Hydrophobic Ligand Entry and Exit Pathways of the CB1 Cannabinoid Receptor

It has been reported that some hydrophobic ligands of G-protein-coupled receptors access the receptor’s binding site from the membrane rather than from bulk water. In order to identify the most probable ligand entrance pathway into the CB1 receptor, we performed several steered molecular dynamics (SMD) simulations of two CB1 agonists, THC and anandamide, pulling them from the receptor’s binding site with constant velocity. The four main directions of ligand pulling were probed: between helices TM4 and TM5, between TM5 and TM6, between TM7 and TM1/TM2, and toward the bulk water. The smallest forces were measured during pulling between TM7 and TM1/TM2. We also performed supervised molecular dynamics (SuMD) simulations for both anandamide and THC entering the CB1 receptor’s binding site and found the same pathway as in the pulling simulations. The residues F174^2.61 and F177^2.64 (both on the TM2 helix) are involved in the gating mechanism and, by forming π–π interactions with ligand molecules, facilitated the ligand orientation required for passage. Using SuMD we also found an alternative binding site for THC. The results of mutagenesis studies evidencing that residues F174^2.61 and F177^2.64 are important for CB1 ligand binding are in agreement with our observations

Hydrophobic Ligand Entry and Exit Pathways of the CB1 Cannabinoid Receptor

It has been reported that some hydrophobic ligands of G-protein-coupled receptors access the receptor’s binding site from the membrane rather than from bulk water. In order to identify the most probable ligand entrance pathway into the CB1 receptor, we performed several steered molecular dynamics (SMD) simulations of two CB1 agonists, THC and anandamide, pulling them from the receptor’s binding site with constant velocity. The four main directions of ligand pulling were probed: between helices TM4 and TM5, between TM5 and TM6, between TM7 and TM1/TM2, and toward the bulk water. The smallest forces were measured during pulling between TM7 and TM1/TM2. We also performed supervised molecular dynamics (SuMD) simulations for both anandamide and THC entering the CB1 receptor’s binding site and found the same pathway as in the pulling simulations. The residues F174^2.61 and F177^2.64 (both on the TM2 helix) are involved in the gating mechanism and, by forming π–π interactions with ligand molecules, facilitated the ligand orientation required for passage. Using SuMD we also found an alternative binding site for THC. The results of mutagenesis studies evidencing that residues F174^2.61 and F177^2.64 are important for CB1 ligand binding are in agreement with our observations

Benchmark results of GPCRM in structure modeling and small molecule docking.

1<p>Here, we provided as a reference results of self-docking to crystal structures of GPCRs.</p>2<p>The binding site area is defined as a set of residues which are inside the 5Å sphere around the ligand.</p

The GPCRM modeling pipeline.

<p>A human intervention is possible in the ‘Advanced’ user mode at the steps indicated by asterisks.</p

Comparison of various methods for the alignment generation in GPCRM.

<p>Here, we plotted ClustalW2 identity scores versus the alignment accuracy (the upper plot) or versus the difference between the accuracy provided by profile-profile alignment and PSA or MSA (the lower plot). The ClustalW2 score and PDB id for both the target and template proteins are provided on the right panel.</p

Antagonist docking to GPCRM-generated homology models versus self-docking: β1AR receptor (A) and D3R receptor (B).

<p>Structures of complexes with indicated polar contacts obtained by crystallography are shown in grey, while the docked structures are depicted in yellow. GPCRM-generated homology models are shown in green. Left panels show the best poses obtained in the docking to corresponding protein homology models. Right panels show results of self-docking to crystallographic structures (PDB id: 2VT4 and 3PBL). All polar contacts were preserved, except one hydrogen bond with Ser211 (A).</p

Towards Improved Quality of GPCR Models by Usage of Multiple Templates and Profile-Profile Comparison

<div><p>G-protein coupled receptors (GPCRs) are targets of nearly one third of the drugs at the current pharmaceutical market. Despite their importance in many cellular processes the crystal structures are available for less than 20 unique GPCRs of the Rhodopsin-like class. Fortunately, even though involved in different signaling cascades, this large group of membrane proteins has preserved a uniform structure comprising seven transmembrane helices that allows quite reliable comparative modeling. Nevertheless, low sequence similarity between the GPCR family members is still a serious obstacle not only in template selection but also in providing theoretical models of acceptable quality. An additional level of difficulty is the prediction of kinks and bulges in transmembrane helices. Usage of multiple templates and generation of alignments based on sequence profiles may increase the rate of success in difficult cases of comparative modeling in which the sequence similarity between GPCRs is exceptionally low. Here, we present GPCRM, a novel method for fast and accurate generation of GPCR models using averaging of multiple template structures and profile-profile comparison. In particular, GPCRM is the first GPCR structure predictor incorporating two distinct loop modeling techniques: Modeller and Rosetta together with the filtering of models based on the Z-coordinate. We tested our approach on all unique GPCR structures determined to date and report its performance in comparison with other computational methods targeting the Rhodopsin-like class. We also provide a database of precomputed GPCR models of the human receptors from that class.</p> <p>Availability</p><p>GPCRM server and database: <a href="http://gpcrm.biomodellab.eu" target="_blank">http://gpcrm.biomodellab.eu</a></p> </div

A scheme of 7TMH fold of Rhodopsin-like class of GPCRs.

<p>Here, we superposed crystal structures of three GPCRs of varied loop conformations: chemokine CXCR4 (PDB id: 3ODU), adrenergic β2AR (2RH1) and adenosine A2AR receptors (2YDV). Except for variety of loop conformations, GPCR structures differ by kinks in TM helices, e.g., in TMH1 (dark blue) and TMH5 (orange), and the length of TM helices, e.g., of TMH7 (dark red).</p

Multiple template modeling of A2AR.

<p>The model (green) was generated by GPCRM and is superposed on the crystal structure (blue) and templates used in the model building: the β1AR adrenergic receptor (grey) and the histamine H1R (pink). The bulge observed in TMH4 in β1AR is properly transferred to the A2AR model. Additionally, incorporation of the second template (H1R) improves the kink of TMH1 in the A2A model. The TMH4 bulge can be examined in details in pictures taken from different angles presented on the left.</p

Comparison of the GPCRM model building procedure based on one, two and three template structures.

1<p>Here, we computed heavy-atoms RMSD of the best model. The binding site area is defined as a set of residues which are in the 5 Å sphere around the ligand in the reference crystal structure.</p>2<p>ClustalW2 scores (normalized to 100) indicating sequence identity are provided in brackets.</p