An Efficient Metadynamics-Based Protocol To Model the Binding Affinity and the Transition State Ensemble of G-Protein-Coupled Receptor Ligands

Abstract

A generally applicable metadynamics scheme for predicting the free-energy profile of ligand binding to G-protein coupled receptors (GPCRs) is described. A common and effective collective variable (CV) has been defined using the ideally placed and highly conserved Trp6.48 as a reference point for ligand-GPCR distance measurement and the common orientation of GPCRs in the cell membrane. Using this single CV together with well-tempered multiple-walker 2 metadynamics with a funnel-like boundary allows an efficient exploration of the entire ligandbinding path from the extracellular medium to the orthosteric binding site, including vestibule and intermediate sites. The protocol can be used with X-ray structures or high-quality homology models for the receptor and is universally applicable to agonists, antagonists, partial and reverse agonists. The root mean square error (RMSE) in predicted binding free energies for 12 diverse ligands in five receptors (a total of 23 data points) is surprisingly small (less than 1 kcal mol − 1 ).The RMSEs for simulations that use receptor X-ray structures and homology models are very similar.

Multiple Binding Sites Contribute to the Mechanism of Mixed Agonistic and Positive Allosteric Modulators of the Cannabinoid CB1 Receptor

“…We recently described am etadynamics-based protocol for studying ligand/GPCR binding and modulation that allows us to study diverse processes from subtype selectivity to the cooperativity between ligand and intracellular binding partner (IBP) and negative allosteric modulation with aroot mean square error (RMSE) between calculated and experimental free energies of binding of less than 1.1 kcal mol À1 and R 2 = 0.74. [34][35][36][37][38] In this work, we built on our previous success in investigating the allostery and cooperativity of GPCR ligands by employing metadynamics simulations to determine binding sites of the agonist CP 55,940 and the Ago-PAM GAT228. We have also determined their binding modes and affinities to delineate the mode of action of 2-phenylindole Ago-PAMs.…”

G-Protein coupled receptors: answers from simulations

“…“Positions” need to be defined in terms of a small number of geometrical variables (the collective variables, CVs) that are relevant (e.g., as a reaction coordinate) for the process being studied. In this respect, the relatively fixed orientation of the GPCR in the membrane allows us to define a generally applicable CV perpendicular to the plane of the membrane [36]. This general CV describes the binding process of ligands approaching from the extracellular medium remarkably well.…”

“…This in itself would not make the simulations effective on massively parallel supercomputers but the use of many replicas at the same time to enhance the sampling (multi-walker metadynamics) [37] further allows many simulations to be carried out in parallel, and thus makes excellent use of massively parallel hardware. The final enhancement to the simulations is to apply a so-called funnel constraint [38] that limits the sampling in the extracellular solution, where it is not necessary [36]. …”

“…This, in turn, allows us to validate the simulations by comparison with experimental free energies of binding obtained from measured binding constants. This comparison turned out to be an unqualified success; the simulated binding energies for 23 different binary and ternary complexes comprising five different receptors and 13 different ligands gave a root mean square deviation of 0.8 kcal mol –1 [36]. In contrast to other techniques used to predict binding energies, the simulations deliver an excellent agreement with the experiment (the correlation line has a slope of 0.99 and an intercept of zero, with R 2 = 0.81), rather than simply correlating well.…”

“…The human chemokine receptor CXCR3, for instance, exhibits distinct alternative binding sites that can be occupied simultaneously by competing ligands, which explains contradictory experimental results obtained in competition experiments [42]. Multiple binding sites have also been found for the β2-adrenergic, muscarinic M2 and μ-opioid receptors [36,39]. …”