Graded activation and free energy landscapes of a muscarinic G-protein–coupled receptor

Abstract

G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M 2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptornanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO-and ARC-bound M 2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M 2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism. cellular signaling | ligand recognition | protein-protein interactions | allostery | drug discovery G -protein-coupled receptors (GPCRs) are primary targets of about one-third of currently marketed drugs. They recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. Increasing experimental and computational evidence suggests that GPCRs exist in an ensemble of different conformations that interconvert dynamically during activation and ligand recognition (1-3). The structure, dynamics, and function of GPCRs result from underlying free energy landscapes (4). However, quantitative characterization of the GPCR activation and ligand-dependent free energy profiles has proved challenging (4-12).The M 2 muscarinic GPCR is widely distributed in mammalian tissues. It plays a key role in regulating the human heart rate and heart contraction forces. The M 2 receptor has been crystallized in both an inactive state bound by the inverse agonist 3-quinuclidinylbenzilate (QNB) (13) and an active state bound by the full agonist iperoxo (IXO) and a G-protein mimetic nanobody (14). The receptor activation is characterized by rearrangements of the transmembrane (TM) helices 5, 6, and 7, particularly closing of the ligan...

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

“…Along this pathway, 24 equally distributed frames-including the crystallographic states-were subjected to conventional MD and GaMD simulations for a total of ∼15 μs with the aim of rigorously characterizing the conformational dynamics of the complex over a long timescale, made possible through the application of enhanced sampling (SI Appendix) (15,16,19). Intermolecular FRET distances have been used to characterize the conformational states of Cas9 during the dynamics ( Fig.…”

“…Here, we characterize the dynamics of the activation pathway of the Cas9 endonuclease through extensive enhanced MD simulations (∼15 μs in length), using a GaMD methodology (18), which allows unconstrained sampling and accurate reconstruction of the free-energy profiles and probes long timescale events over hundreds of microseconds to milliseconds (19,20).…”

“…GaMD has shown robust sampling capability and accurate reconstruction of free-energy profiles for simulations of large biomolecules. It has enabled characterization of ligand binding and conformational changes in G protein-coupled receptors (19) as well as protein folding (20).…”

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

“…Recent simulations on a variety of GPCRs have shown that the extracellular vestibule of the receptor can pre-orient the ligand [34][35][36][37][38][39] and thus provide a well-defined extracellular end-point for docking pathways, and thus simplifying the path-sampling task, often by a form of electrostatic focusing 40 but also by a simple mechanical effect in which part of the ligand is anchored, Additionally, to speed up the sampling, this approach can be easily combined with multiplereplica metadynamics approaches, such as parallel tempering 29 or the multiple-walker technique, 39 which uses several "walkers" to converge one free-energy profile. In our case, we found the trivially parallelizable (and asynchronous) multiple-walker approach more suitable as it is very flexible (the number of replicas can be dynamically changed to use the available resources) and ensures maximum computational efficiency even on non-homogenous clusters.…”

Impact of G protein-coupled receptor heteromers in endocrine systems

“…The explosion of available crystal structures of GPCRs has transformed drug design strategies for the GPCR monomer model, and although crystal structures of homodimers (66)(67) and homo-oligomers (68) have been identified, heteromeric structures would provide unprecedented information, not only on interfaces, but unique heteromer-specific targeting sites for structure-based drug discovery. With the generation of antibodies that target GPCR heteromers and the use of nanobodies in GPCR research (69)(70)(71)(72)(73)(74)(75)(76), there is a real technical possibility of creating tools to aid stabilisation of such structures for crystallization. Likewise there is increase interested in immunotherapy pathways, particularly in cancer, and whilst there are no current approved GPCR antibody drugs, antibodies specific to GPCR heteromers have been generated (77).…”