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

Abstract

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

CCR5 adopts three homodimeric conformations that control cell surface delivery

“…2B and 3E). The multiplicity of unliganded forms of CCR5 is consistent with previous computational analyses that predicted the existence of several apo forms of CCR5 with different stabilities (33). The coexistence of several dimeric conformations is also consistent with a single-molecule fluorescence imaging study that showed preformed diverse dimeric structures of another class A GPCR (11).…”

“…Small-molecular weight ligands can act as pharmacological chaperones by rescuing the delivery of poorly trafficked, disease-causing GPCRs (31,32). We tested the effect of MVC, which is a partial inverse agonist of CCR5 (33,34), on the cell surface delivery of CCR5 dimerizationcompromised mutants. We hypothesized that this compound might stabilize a conformation of the receptors suitable for export from the ER.…”

The Predicted Ensemble of Low‐Energy Conformations of Human Somatostatin Receptor Subtype 5 and the Binding of Antagonists

“…To predict the 3D structures of hSSTR5, we developed a modified version of G PCR En semble of S tructures in M embrane B i L ayer E nvironment (GEnSeMBLE)10 complete sampling computational method, which aims to predict the 10 to 25 lower‐energy protein structures likely to play a role in activation upon binding various ligands. GEnSeMBLE has been successfully applied in the prediction of inactive‐state structures of GPCRs such as cannabinoid receptor type 1 (CB1),8a adenosine A3 receptor (hAA 3 R),8c olfactory receptor OR1G1,8d bitter taste receptor Tas2R38,8e the GLP‐1 receptor (GLP1R),8f and C‐C chemokine receptor type 5 (CCR5) 8g. For CB1 and hAA 3 R, the active states were also identified successfully.…”

mutLBSgeneDB: mutated ligand binding site gene DataBase

“…Mutation-induced conformational change and induced fit with the ligand are the key factors of protein–ligand interactions in cancer cells (2,3). Point mutations at spatially distinct sites lead to conformational changes and exert hinge effects (4).…”