Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor

Abstract

Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs.

Keywords: G-protein coupled receptor; Ligand binding; M3 muscarinic receptor; accelerated molecular dynamics; enhanced sampling.

Figure 1

(a) Schematic representation of the M3 muscarinic receptor-ligand binding simulation system and (b) three known ligands of the M3 receptor that are selected for aMD simulations: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh).

Figure 2

(a) Schematic representation of the X-ray crystal structure of the M3 muscarinic receptor bound to the antagonist, tiotropium (TTP). With TTP removed, this structure was used for aMD simulations of the binding of three known ligand molecules: antagonist TTP, partial agonist arecoline (ARc), and full agonist acetylcholine (ACh). (b) RMSDs are plotted for the heavy atoms of each ligand relative to the crystal structure (for TTP) or the top-ranked docking pose (for ARc and ACh) in the orthosteric binding site after aligning all simulation frames using the C_α atoms of the receptor transmembrane bundle. Here, data is shown only for ligand molecules that bound to the receptor at some point during the simulations (see Fig. S1 for RMSDs of all ligands). Note that in the TTP RMSD plot the blue and black traces represent two different ligand molecules in one simulation, while in the ACh RMSD plot the two curves represent ligand molecules in two different simulations. Only one ARc molecule bound to the receptor, thus there is just a single curve in the ARc RMSD plot. In all cases, the ligand is bound to the receptor at RMSD values less than approximately 20 Å.

Figure 3

(a) The three most populated TTP binding clusters are located in the extracellular vestibule and are shown in blue, gray, and purple, respectively. (b) Key residues interacting with TTP in cluster A (blue) are shown in sticks and the representative receptor structure observed in the aMD simulations is shown in blue ribbons. A full list of contact residues is given in Table S3.

Figure 4

(a) The three most populated ACh binding clusters are shown in purple, blue and gray, respectively. (b) Trajectory of ACh diffusing between the three clusters during a 200 ns aMD simulation. The time evolution for the other 200ns aMD simulation in which ACh bound to the receptor is plotted in Fig. S3. Key residues in contact with ACh are shown for the (c) Cluster C, (d) Cluster B, and (e) Cluster A. A full list of contact residues is given in Table S3.

Figure 5

(a) The three most populated ARc binding clusters are shown in blue, purple, and gray, respectively. (b) Trajectory of ARc diffusing between the three clusters during a 200ns aMD simulation in which binding occurred. Key residues interacting with ARc are shown for (c) Cluster C, (d) Cluster B, and (e) Cluster A. A full list of contact residues is given in Table S3.