Probe Confined Dynamic Mapping for G Protein-Coupled Receptor Allosteric Site Prediction.
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ABSTRACT:
SUBMITTER: Ciancetta A
PROVIDER: S-EPMC8614102 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and ?-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the ?2-adrenergic receptor (?2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct ?2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for ?2AR in the presence of Nb80 compared to the affinity of isoprenaline for ?2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the ?2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.
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Project description:The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [(3)H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of ORG27569 at CB1. To this end, we used computation, synthesis, mutation, and functional studies to identify the ORG27569-binding site in the CB1 TMH3-6-7 region. This site is consistent with the results of K3.28(192)A, F3.36(200)A, W5.43(279)A, W6.48(356)A, and F3.25(189)A mutation studies, which revealed the ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28(192) that is important for ORG27569 to act as an inverse agonist. At this allosteric site, ORG27569 promotes an intermediate conformation of the CB1 receptor, explaining ORG27569's ability to increase equilibrium binding of CP55,940. This site also explains ORG27569's ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63(176). 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.
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Project description:Metabotropic glutamate receptors (mGluR) are mainly expressed in the central nervous system (CNS) and contain eight receptor subtypes, named mGluR1 to mGluR8. The crystal structures of mGluR1 and mGluR5 that are bound with the negative allosteric modulator (NAM) were reported recently. These structures provide a basic model for all class C of G-protein coupled receptors (GPCRs) and may aid in the design of new allosteric modulators for the treatment of CNS disorders. However, these structures are only combined with NAMs in the previous reports. The conformations that are bound with positive allosteric modulator (PAM) or agonist of mGluR1/5 remain unknown. Moreover, the structural information of the other six mGluRs and the comparisons of the mGluRs family have not been explored in terms of their binding pockets, the binding modes of different compounds, and important binding residues. With these crystal structures as the starting point, we built 3D structural models for six mGluRs by using homology modeling and molecular dynamics (MD) simulations. We systematically compared their allosteric binding sites/pockets, the important residues, and the selective residues by using a series of comparable dockings with both the NAM and the PAM. Our results show that several residues played important roles for the receptors' selectivity. The observations of detailed interactions between compounds and their correspondent receptors are congruent with the specificity and potency of derivatives or compounds bioassayed in vitro. We then carried out 100 ns MD simulations of mGluR5 (residue 26-832, formed by Venus Flytrap domain, a so-called cysteine-rich domain, and 7 trans-membrane domains) bound with antagonist/NAM and with agonist/PAM. Our results show that both the NAM and the PAM seemed stable in class C GPCRs during the MD. However, the movements of "ionic lock," of trans-membrane domains, and of some activation-related residues in 7 trans-membrane domains of mGluR5 were congruent with the findings in class A GPCRs. Finally, we selected nine representative bound structures to perform 30 ns MD simulations for validating the stabilities of interactions, respectively. All these bound structures kept stable during the MD simulations, indicating that the binding poses in this present work are reasonable. We provided new insight into better understanding of the structural and functional roles of the mGluRs family and facilitated the future structure-based design of novel ligands of mGluRs family with therapeutic potential.
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Project description:One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTP?S and ?-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTP?S assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for ?-arrestin1 recruitment, PLC?3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space.
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Project description:We developed a general cell-based photocrosslinking approach to investigate the binding interfaces necessary for the formation of G protein-coupled receptor (GPCR) signaling complexes. The two photoactivatable unnatural amino acids p-benzoyl-L-phenylalanine and p-azido-L-phenylalanine were incorporated by amber codon suppression technology into CXC chemokine receptor 4 (CXCR4). We then probed the ligand-binding site for the HIV-1 coreceptor blocker, T140, using a fluorescein-labeled T140 analogue. Among eight amino acid positions tested, we found a unique UV-light-dependent crosslink specifically between residue 189 and T140. These results are evaluated with molecular modeling using the crystal structure of CXCR4 bound to CVX15.
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Project description:G protein-coupled receptors (GPCRs) are the most common targets of drug discovery. However, the similarity between related GPCRs combined with the complex spatiotemporal dynamics of receptor activation in vivo has hindered drug development. Photopharmacology offers the possibility of using light to control the location and timing of drug action by incorporating a photoisomerizable azobenzene into a GPCR ligand, enabling rapid and reversible switching between an inactive and active configuration. Recent advances in this area include (i) photoagonists and photoantagonists that directly control receptor activity but are nonselective because they bind conserved sites, and (ii) photoallosteric modulators that bind selectively to nonconserved sites but indirectly control receptor activity by modulating the response to endogenous ligand. In this study, we designed a photoswitchable allosteric agonist that targets a nonconserved allosteric site for selectivity and activates the receptor on its own to provide direct control. This work culminated in the development of aBINA, a photoswitchable allosteric agonist that selectively activates the Gi/o-coupled metabotropic glutamate receptor 2 (mGluR2). aBINA is the first example of a new class of precision drugs for GPCRs and other clinically important signaling proteins.
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Project description:G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.
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Project description:G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active ?(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.
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