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DEER Analysis of GPCR Conformational Heterogeneity

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DEER Analysis of GPCR Conformational Heterogeneity biomolecules Review DEER Analysis of GPCR Conformational Heterogeneity Matthias Elgeti * and Wayne L. Hubbell * Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA * Correspondence: [email protected] (M.E.); [email protected] (W.L.H.); Tel.: +1-(310)-206-8831 (M.E. & W.L.H.) 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. Citation: Elgeti, M.; Hubbell, W.L. Keywords: G protein-coupled receptor; GPCR; 7TM receptor; G protein; arrestin; structure; function; DEER Analysis of GPCR structural plasticity; electron paramagnetic resonance; EPR; DEER; pELDOR Conformational Heterogeneity. Biomolecules 2021, 11, 778. https:// doi.org/10.3390/biom11060778 1. Introduction Academic Editor: Karsten Melcher G protein-coupled receptors (GPCRs), also known as seven-transmembrane (7TM) receptors, represent the largest class of transmembrane proteins in the human genome Received: 1 May 2021 (>800 members, [1]). Despite their homologous 7TM fold (Figure1a), different GPCRs may Accepted: 19 May 2021 bind to a large variety of different ligands, and each ligand/GPCR pair exhibits character- Published: 22 May 2021 istic pharmacological properties such as affinity, efficacy, potency and signal bias. On the intracellular side, GPCRs couple to a relatively small set of transducer proteins such as G Publisher’s Note: MDPI stays neutral proteins, GPCR kinases (GRKs) and arrestins, which, once activated, bind and modulate the with regard to jurisdictional claims in activity of downstream effector proteins and thus signaling pathways (Figure1b). Hence, published maps and institutional affil- iations. GPCRs function as promiscuous and yet highly specific signaling proteins (“allosteric microprocessor” [2]) channeling the binding events of distinct ligands towards different and specific physiological outcomes. This finding has been conceptualized in terms of a conformational selection model: a manifold of distinct receptor conformations coexist in equilibrium, each exhibiting differences in the orientation of structural elements, which Copyright: © 2021 by the authors. range from individual amino acids side chains to secondary and tertiary structures. These Licensee MDPI, Basel, Switzerland. differences translate into distinct affinities and catalytic activities of each conformation This article is an open access article towards intracellular transducer proteins. By binding and stabilizing specific receptor con- distributed under the terms and formations, ligands modulate transducer interactions and trigger a characteristic cellular conditions of the Creative Commons Attribution (CC BY) license (https:// signaling response (Figure1b). In this simple framework, basal activity is achieved by creativecommons.org/licenses/by/ residual amounts of an active conformation [3]. Biased signaling (also known as functional 4.0/). selectivity) is the ability of ligands to tune signaling towards a specific transducer protein, Biomolecules 2021, 11, 778. https://doi.org/10.3390/biom11060778 https://www.mdpi.com/journal/biomolecules BiomoleculesBiomolecules 20212021, ,1111, ,x 778 2 of2 of22 21 protein,and may and be may understood be understood as an equilibrium as an equili shiftbrium between shift between active conformationsactive conformations of distinct of distinctaffinity/catalytic affinity/catalytic activity activity towards towards a specific a transducer.specific transducer. Traditionally, Traditionally, a ligand’s a signal ligand’s bias signalis quantified bias is byquantified comparing by thecomparing cellular signalingthe cellular responses signaling to thatresponses of a “balanced” to that of reference, a “bal- anced”such as reference, the endogenous such as the ligand endogenous [4]. From lig aand biochemical [4]. From standpoint, a biochemical ligand standpoint, binding lig- and andtransducer binding couplingand transducer to a GPCR coupling may beto a treated GPCR equally; may be eachtreated binding equally; event each stabilizes binding a eventspecific stabilizes GPCR a conformational specific GPCR conformational state, thereby modulating state, thereby the modulating affinity towards the affinity the other to- wards(ternary the complex other (ternary [5–7]). complex [5–7]). FigureFigure 1. 1. GPCRGPCR function function seen seen from from the the DEER DEER vantage vantage point. point. (a ()a Structural) Structural homology homology among among GPCRs GPCRs of of various various functions. functions. Receptor-specificReceptor-specific ligands ligands bind bind from from the extracellular side, which therefore showsshows highhigh structuralstructural diversity diversity between between different differ- entreceptors. receptors. The The transmembrane transmembrane core core exhibits exhibits many many conservedconserved interactioninteraction networks,networks, suggesting a a common common activation activation mechanism.mechanism. On On the the intracellular intracellular side, side, a ali limitedmited set set of of transducer transducer proteins proteins (such (such as as G G protein, protein, arrestin arrestin and and GRK) GRK) bind bind to to specificspecific receptor receptor conformations. conformations. (b (b) )The The GPCR GPCR “allosteric “allosteric microprocessor”. microprocessor”. Bindin Bindingg of of an an extracellular extracellular ligand ligand is is translated translated intointo ligand- ligand- and and receptor-specific receptor-specific affiniti affinitieses and and catalytic catalytic activities activities towards towards transd transducerucer proteins. proteins. Activated Activated transducer transducer pro- teinsproteins interact interact with with different different effectors effectors and and ther therebyeby elicit elicit a ligand-specific a ligand-specific cellular cellular response. response. (c ()c )Conformational Conformational selection selection from the DEER perspective. Different ligand classes (such as antagonists, balanced/reference and biased agonists) stabilize 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 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 absencepopulations of ligand. are encoded as distance distributions derived from DEER. Note that all conformations are present even in the absence of ligand. In order to characterize ligand action, for example during drug design, the structural In order to characterize ligand action, for example during drug design, the structural differences of functionally distinct receptor conformations and their equilibrium popula- differences of functionally distinct receptor conformations and their equilibrium popula- tions need to be determined. These characteristics may then be directly related to respec- tions need to be determined. These characteristics may then be directly related to respective tive ligand parameters such as affinity, bias and efficacy (the propensity of a ligand to ligand parameters such as affinity, bias and efficacy (the propensity of a ligand to elicit a elicit a physiological response). Mapping the complex conformational landscape of physiological response). Mapping the complex conformational landscape of GPCRs is a GPCRs is a challenge for methods such as X-ray diffraction, wherein the receptor is typi- challenge for methods such as X-ray diffraction, wherein the receptor is typically removed cally removed from its native bilayer environment by solubilization in detergents and from its native bilayer environment by solubilization in detergents and crystallized in a crystallizedlattice where in intermoleculara lattice where forcesintermolecular stabilize aforces unique stabilize conformation. a unique Stabilizing conformation. mutations Sta- bilizingor crystallization mutations “helpers”or crystallization are often “helpers required” toare facilitate often required crystallization to facilitate [8–10
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