Computationally‐predicted CB1 cannabinoid receptor mutants show distinct patterns of salt‐bridges that correlate with their level of constitutive activity reflected in G protein coupling levels, thermal stability, and ligand binding

Ahn, Kyu‐Hong; Scott, Caitlin E.; Abrol, Ravinder; Goddard, William A.; Kendall, Debra A.

doi:10.1002/prot.24264

Cited by 34 publications

(42 citation statements)

References 84 publications

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“…Indeed functional differences between agonists and inverse agonists (or antagonists) to GPCRs are well established. For example, recent studies show that single, double, and triple mutant forms of the CB1 receptor exhibit dramatic changes in function, switching from full inactivity to high constitutive activity and back for sequential mutations (26,27). (iv) The GEnSeMBLE approach for predicting the ensemble of thermally accessible protein-ligand complexes provides a tool for accurate GPCR-ligand predictions consistent with the GPCR pleiotropy paradigm.…”

Section: Discussionmentioning

confidence: 99%

Ligand- and mutation-induced conformational selection in the CCR5 chemokine G protein-coupled receptor

Abrol

Trzaskowski

Goddard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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We predicted the structural basis for pleiotropic signaling of the C-C chemokine type 5 (CCR5) G protein-coupled receptor (GPCR) by predicting the binding of several ligands to the lower-energy conformations of the CCR5 receptor and 11 mutants. For each case, we predicted the ∼20 most stable conformations for the receptor along with the binding sites for four anti-HIV ligands. We found that none of the ligands bind to the lowest-energy apo-receptor conformation. The three ligands with a similar pharmacophore (Maraviroc, PF-232798, and Aplaviroc) bind to a specific higher-energy receptor conformation whereas TAK-779 (with a different pharmacophore) binds to a different high-energy conformation. This result is in agreement with the very different binding-site profiles for these ligands obtained by us and others. The predicted Maraviroc binding site agrees with the recent structure of CCR5 receptor cocrystallized with Maraviroc. We performed 11 site-directed mutagenesis experiments to validate the predicted binding sites. Here, we independently predicted the lowest 10 mutant protein conformations for each of the 11 mutants and then docked the ligands to these lowest conformations. We found the predicted binding energies to be in excellent agreement with our mutagenesis experiments. These results show that, for GPCRs, each ligand can stabilize a different protein conformation, complicating the use of cocrystallized structures for ligand screening. Moreover, these results show that a single-point mutation in a GPCR can dramatically alter the available low-energy conformations, which in turn alters the binding site, potentially altering downstream signaling events. These studies validate the conformational selection paradigm for the pleiotropic function and structural plasticity of GPCRs.protein structure prediction | ligand-protein binding prediction | conformational ensemble | functional selectivity | GEnSeMBLE

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Section: Discussionmentioning

confidence: 99%

Ligand- and mutation-induced conformational selection in the CCR5 chemokine G protein-coupled receptor

Abrol

Trzaskowski

Goddard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition to CB1 (7,8), SuperBiHelix has been applied successfully to the adenosine A 3 receptor (16) and other adenosine receptors (22), serotonin 5-HT2B and 5-HT2C receptor (25), the histamine H3 receptor (26), the CCR5 receptor (27,28), TAS2R38 bitter taste receptor (29), and the V2 vasopressin receptor (30). The predicted GPCR structures in these studies were validated by predicting the binding sites and energies for known series of ligands and comparing with experimental mutagenesis, binding, and/or functional data.…”

Section: Discussionmentioning

confidence: 99%

“…The structures lead to an activation mechanism for CB1 that explains all experiments and that may play a role in other GPCRs. Additional mutants have also been designed and then tested in GTPγS assays (8), lending strong support to this activation mechanism.…”

Section: Validationmentioning

confidence: 96%

SuperBiHelix method for predicting the pleiotropic ensemble of G-protein–coupled receptor conformations

Bray

Abrol

Goddard

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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There is overwhelming evidence that G-protein-coupled receptors (GPCRs) exhibit several distinct low-energy conformations, each of which might favor binding to different ligands and/or lead to different downstream functions. Understanding the function of such proteins requires knowledge of the ensemble of low-energy configurations that might play a role in this pleiotropic functionality. We earlier reported the BiHelix method for efficiently sampling the (12) 7 = 35 million conformations resulting from 30°rotations about the axis (η) of all seven transmembrane helices (TMHs), showing that the experimental structure is reliably selected as the best conformation from this ensemble. However, various GPCRs differ sufficiently in the tilts of the TMHs that this method need not predict the optimum conformation starting from any other template. In this paper, we introduce the SuperBiHelix method in which the tilt angles (θ, φ) are optimized simultaneously with rotations (η) efficiently enough that it is practical and sufficient to sample (5 × 3 × 5) 7 = 13 trillion configurations. This method can correctly identify the optimum structure of a GPCR starting with the template from a different GPCR. We have validated this method by predicting known crystal structure conformations starting from the template of a different protein structure. We find that the SuperBiHelix conformational ensemble includes the higher energy conformations associated with the active protein in addition to those associated with the more stable inactive protein. This methodology was then applied to design and experimentally confirm structures of three mutants of the CB1 cannabinoid receptor associated with different functions.protein structure prediction | A 2A adenosine receptor | β 2 -adrenergic receptor | constitutive activity | functional selectivity

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“…Accordingly, a number of studies have aimed to identify such regulatory motifs of CB 1 R. A detailed computational model based on the crystal structure of the b 2 -adrenergic receptor-Ga s complex, combined with mutational data, suggested that distinct residues in the ICL2 and ICL3 regions of the CB 1 R may be involved in the stabilization of the active, Ga i -coupled receptor conformation (Shim et al 2013). Two other recent studies have analyzed the role of several intramolecular salt-bridges, which may stabilize inactive, partially active, and fully active CB 1 R conformations (Ahn et al 2013b, Scott et al 2013. According to this model, D3.49 and R3.50 residues form salt-bridges with K4.41 and D6.30, respectively, which (together with a D2.63CK3.28 salt-bridge) may keep the receptor in a partially active conformation under basal conditions.…”

Section: Introductionmentioning

confidence: 99%

Mutations in the ‘DRY’ motif of the CB1 cannabinoid receptor result in biased receptor variants

Gyombolai¹,

Tóth²,

Tímár³

et al. 2014

Journal of Molecular Endocrinology

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The role of the highly conserved 'DRY' motif in the signaling of the CB 1 cannabinoid receptor (CB 1 R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB 1 R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric G o proteins (w80% of WT CB 1 R (CB 1 R-WT)). Moreover, this mutant showed an enhanced basal b-arrestin2 (b-arr2) recruitment. More strikingly, the double-mutant CB 1 R-D3.49A/R3.50A was biased toward b-arrs, as it gained a robustly increased b-arr1 and b-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB 1 R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit b-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (w70% of CB 1 R-WT). Agonist-induced ERK1/2 activation of the CB 1 R mutants showed a good correlation with their b-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and b-arr binding of the CB 1 R are mediated by distinct receptor conformations, and the conserved 'DRY' motif plays different roles in the stabilization of these conformations, thus mediating both G-protein-and b-arr-mediated functions of CB 1 R.

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Computationally‐predicted CB1 cannabinoid receptor mutants show distinct patterns of salt‐bridges that correlate with their level of constitutive activity reflected in G protein coupling levels, thermal stability, and ligand binding

Cited by 34 publications

References 84 publications

Ligand- and mutation-induced conformational selection in the CCR5 chemokine G protein-coupled receptor

Ligand- and mutation-induced conformational selection in the CCR5 chemokine G protein-coupled receptor

SuperBiHelix method for predicting the pleiotropic ensemble of G-protein–coupled receptor conformations

Mutations in the ‘DRY’ motif of the CB1 cannabinoid receptor result in biased receptor variants

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