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.

Keywords: 7TM receptor; DEER; EPR; G protein; G protein-coupled receptor; GPCR; arrestin; electron paramagnetic resonance; function; pELDOR; structural plasticity; structure.

Figure 1

GPCR function seen from the DEER vantage point. (a) Structural homology among GPCRs of various functions. Receptor-specific ligands bind from the extracellular side, which therefore shows high structural diversity between different receptors. The transmembrane core exhibits many conserved interaction networks, suggesting a common activation mechanism. On the intracellular side, a limited set of transducer proteins (such as G protein, arrestin and GRK) bind to specific receptor conformations. (b) The GPCR “allosteric microprocessor”. Binding of an extracellular ligand is translated into ligand- and receptor-specific affinities and catalytic activities towards transducer proteins. Activated transducer proteins interact with different effectors and thereby elicit a ligand-specific cellular response. (c) Conformational selection from the DEER perspective. Different ligand classes (such as antagonists, balanced/reference and biased agonists) stabilize distinct GPCR conformations, leading to specific efficacies towards transducers. The different conformations and their populations are encoded as distance distributions derived from DEER. Note that all conformations are present even in the absence of ligand.

Figure 2

GPCR conformational heterogeneity. Distance distributions between spin labels attached to the indicated receptor segments reveal conformational heterogeneity of AT1R, which is differentially modulated by ligands of distinct function (in italics). Ang II is the endogenous, balanced agonist, while TRV026/034 and TRV055 represent β-arrestin and G protein-biased ligands, respectively. Dotted lines indicate main conformations. For this study, the cytoplasmic ends of AT1R TMs were spin labeled using IDSL (cf. [38] for the full dataset).

Figure 3

NNMF analysis. (a) NNMF analysis of AT1R DEER data reveals four major conformational states C1–C4 (columns of matrix C). Residual multimodality in each conformation suggests Table 1. C4 under the various ligand conditions. Bias of different agonists can be identified by the population of C2, C3 and C4.

Figure 4

DEER mapping. Cytoplasmic surface of AT1R with spin label side chains modeled indicating the reference sites, TM1 and ICL2, and the monitor site TM6. Shown spin densities were calculated from main NNMF distances via 2D trilateration. For TM6^C4, the used distances d^C4 and intersecting spheres (dotted lines) are shown for visualization. The underlying structure was derived from MD simulations of antagonist bound AT1R [38].

Figure 5

Spin labels commonly used for DEER. (a) MTSL: Reaction with the thiol side chain creates the disulfide-linked side chain R1. (b) IAP: S_N2 reaction creates the thioether-linked PROXYL side chain. (c) IDSL: Sulfhydryl-disulfide exchange leads to formation of the short spin label side chain V1.