Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of ...arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: 'key sites' required for arrestin binding and activation, an 'inhibitory site' that abrogates arrestin binding, and 'modulator sites' that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.
•Recent technological advances in GPCR structural biology have led to many new and exciting structures.•Protein engineering remains important for determining new structures.•Advances in XFEL and ...synchrotron technology offer new methods for data collection.•Multiple active-state GPCR-G protein bound structures have been determined by cryo-EM.
G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface receptor proteins and are important drug targets for many human diseases. In the last decade, remarkable progress has been made in the determination of atomic structures of GPCRs with over 200 structures from 53 unique receptors having been solved. Technological advances in protein engineering and X-ray crystallography have driven much of the progress to date. However, recent advances in cryo-electron microscopy have facilitated the structural determination of three new structures of active-state GPCRs in complex with heterotrimeric G protein. These advances have led to significant breakthroughs in our understanding of GPCR biology including not only how signal transducers such as G proteins or arrestins interact with receptors, but also pave the way for future structure-based drug design.
Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern ...the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M
muscarinic acetylcholine receptor (M
mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M
mAChR. By determining the cryo-electron microscopy structures of the M
mAChR, bound to a cognate G
protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.
Abstract
The M
4
muscarinic acetylcholine receptor (M
4
mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of ...psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M
4
mAChR in complex with the heterotrimeric G
i1
transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M
4
mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M
4
mAChR. These findings provide a basis for further understanding xanomeline’s complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.
G protein-coupled receptors (GPCRs) are flexible integral membrane proteins involved in transmembrane signaling. Their involvement in many physiological processes makes them interesting targets for ...drug development. Determination of the structure of these receptors will help to design more specific drugs, however, their structural characterization has so far been hampered by the low expression and their inherent instability in detergents which made protein engineering indispensable for structural and biophysical characterization. Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination. These include truncations of the flexible regions, stabilization by antibodies and nanobodies, fusion partners, high affinity and covalently bound ligands as well as conformational stabilization by mutagenesis. In this review we focus on stabilization of GPCRs by insertion of point mutations, which lead to increased conformational and thermal stability as well as improved expression levels. We summarize existing mutagenesis strategies with different coverage of GPCR sequence space and depth of information, design and transferability of mutations and the molecular basis for stabilization. We also discuss whether mutations alter the structure and pharmacological properties of GPCRs.
The human M₅ muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of ...structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M₅ mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M₂ and M₅ mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.
In the absence of orthosteric ligands, most G protein-coupled receptors (GPCRs) exist in an equilibrium of different conformational states. This equilibrium is shifted by an agonist towards the ...active state or by an inverse agonist towards the inactive state. The basal activity of the receptor, and its ability to activate intracellular signaling pathways, is defined by the probability that a fraction of the receptor adopts the active state in the absence of ligand. Despite breakthroughs in native MS of membrane proteins, GPCR-transducing complexes have not been studied by this approach until very recently. Here, we investigated different conformational states of the turkey β1 adrenergic receptor (tβ1AR) in complex with two transducing partners: a G protein mimicking nanobody, Nb80, and an engineered truncated Gs protein (miniG
s
), in the presence of the full agonist isoprenaline by native MS. Interestingly, complex formation with both transducing partners was also observed in the absence of agonist, and allowed us to quantify basal activity of tβ1AR. We followed the stepwise disassembly of the transducing complexes by increasing the concentration of the inverse agonist S32212 in the presence of a constant concentration of isoprenaline. This allowed us to determine the relative binding affinity of S32212 in comparison to isoprenaline by native MS. Our approach provides a fast and sensitive way to detect complexes, study their stability in the presence of different ligands, and determine relative ligand affinities. Native mass spectrometry thus has the potential to become a useful tool to screen for orthosteric and allosteric GPCR drugs.
Graphical Abstract
GPCR Solubilization and Quality Control Miljus, Tamara; Sykes, David A; Harwood, Clare R ...
Methods in molecular biology (Clifton, N.J.),
01/2020, Letnik:
2127
Journal Article
G protein-coupled receptors (GPCRs) are versatile membrane proteins involved in the regulation of many physiological processes and pathological conditions, making them interesting pharmacological ...targets. In order to study their structure and function, GPCRs are traditionally extracted from membranes using detergents. However, due to their hydrophobic nature, intrinsic instability in aqueous solutions, and their denaturing effects, the isolation of properly folded and functional GPCRs is not trivial. Therefore, it is of crucial importance to solubilize receptors under mild conditions and control the sample quality subsequently. Here we describe widely used methods for small-scale GPCR solubilization, followed by quality control based on fluorescence size-exclusion chromatography, SDS-PAGE, temperature-induced protein unfolding (CPM dye binding) and fluorescent ligand binding assay. These methods can easily be used to assess the thermostability and functionality of a GPCR sample exposed to different conditions, such as the use of various detergents, addition of lipids and ligands, making them valuable for obtaining an optimal sample quality for structural and functional studies.
The human M
muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural ...information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M
mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M
and M
mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.
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