DEER Analysis of GPCR Conformational Heterogeneity

Abstract

G protein-coupled receptors (GPCRs) represent a large class of transmembrane helical proteins which are involved in numerous physiological signaling pathways and therefore represent crucial pharmacological targets. GPCR function and the action of therapeutic molecules are defined by only a few parameters, including receptor basal activity, ligand affinity, intrinsic efficacy and signal bias. These parameters are encoded in characteristic receptor conformations existing in equilibrium and their populations, which are thus of paramount interest for the understanding of receptor (mal-)functions and rational design of improved therapeutics. To this end, the combination of site-directed spin labeling and EPR spectroscopy, in particular double electron–electron resonance (DEER), is exceedingly valuable as it has access to sub-Angstrom spatial resolution and provides a detailed picture of the number and populations of conformations in equilibrium. This review gives an overview of existing DEER studies on GPCRs with a focus on the delineation of structure/function frameworks, highlighting recent developments in data analysis and visualization. We introduce “conformational efficacy” as a parameter to describe ligand-specific shifts in the conformational equilibrium, taking into account the loose coupling between receptor segments observed for different GPCRs using DEER.

Insights into the Promising Prospect of G Protein and GPCR-Mediated Signaling in Neuropathophysiology and Its Therapeutic Regulation

“…Studies conducted with partial or complete agonists demonstrated wide variations in their activities, especially in weaker patients [ 34 ]. However, these findings highlighted several uncertainties, as the ligands involved here were bound to noradrenergic imidazoline receptors that were present on platelets [ 35 , 36 , 37 ]. Considering these findings, the sensitivity of alpha2 receptors seems inconclusive.…”

Metadynamics simulations leveraged by statistical analyses and artificial intelligence-based tools to inform the discovery of G protein-coupled receptor ligands

“…To probe the conformational heterogeneity of GPCRs and GPCR complexes and extend information from highresolution structural methods, researchers have resorted to biophysical techniques such as nuclear magnetic resonance (14-21), double electron-electron resonance spectroscopy (22), hydrogen/deuterium exchange mass spectroscopy (23, 24), and single-molecule fluorescence resonance energy transfer (25)(26)(27). However, current technical challenges prevent these techniques from achieving atomic-level precision for the entire GPCR alone or in complex with their natural cellular signaling partners.…”

Thermodynamic Architecture and Conformational Plasticity of GPCRs

“…Aside from all-atom MD simulations, [9][10][11][12][13][14][15] multiple biophysical techniques have been used to probe GPCR conformational dynamics, ligandbinding, and GPCR-G-protein interactions at timescales of nanoseconds to seconds. These include Förster resonance energy transfer (FRET), [16][17][18][19][20] hydrogen/deuterium exchange mass spectrometry (HDX-MS), [21][22][23][24][25] electron paramagnetic resonance (EPR) spectroscopy, [26][27][28][29][30] and nuclear magnetic resonance (NMR) spectroscopy. [31][32][33][34] Alongside static structures of GPCRs in unbound, ligandbound, and transducer-bound states, these experiments have revealed the helix movements that occur upon GPCR activation, specific structural features that mediate these movements and the possibility of intermediates during the transition between active and inactive conformations.…”