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Agonist binding by the β2-adrenergic receptor: an effect of receptor conformation on ligand association-dissociation characteristics.

ABSTRACT: The β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR), is a physiologically important transmembrane protein that is a target for drugs used for treatment of asthma and cardiovascular diseases. Study of the first steps of ligand recognition and the molecular basis of ligand binding to the orthosteric site is essential for understanding the pharmacological properties of the receptor. In this work we investigated the characteristic features of the agonist association-dissociation process to and from the different conformational forms of β2-AR by use of advanced molecular modeling techniques. The investigation was focused on estimating the free energy profiles (FEPs) corresponding to the process of a full agonist ((R,R)-fenoterol) and an inverse agonist (carazolol) binding and unbinding to and from β2-AR. The two different conformational forms of β2-AR, i.e. active β2-AR-PDB: 3P0G and inactive β2-AR-PDB: 2RH1 were included in this stage of the study. We revealed several significant qualitative differences between FEPs characteristic of both conformational forms. Both FEPs suggest the existence of three transient binding sites in the extracellular domain of β2-AR. Comparison of the residues surrounding these transient binding sites in both β2-AR states revealed the importance of the aromatic residues F194, H93(2.64), H296(6.58), and H178 (extracellular part of β2-AR) in the early stages of the binding process. In addition, slightly different exit and entry paths are preferred by the ligand molecule in the extracellular part of β2-AR, depending on the conformation of the receptor.

SUBMITTER: Plazinska A

PROVIDER: S-EPMC4359354 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nanobody binding stabilizes G-protein-coupled receptors (GPCR) in a fully active state and modulates their affinity for bound ligands. However, the atomic-level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like β2 adrenergic receptor (β2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes structural features of the β2AR with an ∼14 Å outward movement of transmembrane helix 6 and a close proximity of transmembrane (TM) helices 5 and 7, and favors the fully active-like conformation of the receptor, independent of ligand binding, in contrast to the conditions under which no intracellular binding partner is bound, in which case the receptor is only stabilized in an intermediate-active state. This activation is supported by the residues located at hotspots located on TMs 5, 6, and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intracellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding triggers tighter and stronger local communication networks between the Nb80 and the ligand-binding sites, primarily involving residues around ICL2 and the intracellular end of TM3, TM5, TM6, as well as ECL2, ECL3, and the extracellular ends of TM6 and TM7. In particular, we identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in conformations modulated by a full agonist, BI167107, and a G-protein-biased partial agonist, salmeterol, involving mainly TM1 and TM2, and TM5, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which should be taken into account in structure-based drug discovery.

Project description:G protein-coupled receptors are involved in many biological processes, relaying the extracellular signal inside the cell. Signaling is regulated by the interactions between receptors and their ligands, it can be stimulated by agonists, or inhibited by antagonists or inverse agonists. The development of a new drug targeting a member of this family requires to take into account the pharmacological profile of the designed ligands in order to elicit the desired response. The structure-based virtual screening of chemical libraries may prioritize a specific class of ligands by combining docking results and ligand binding information provided by crystallographic structures. The performance of the method depends on the relevance of the structural data, in particular the conformation of the targeted site, the binding mode of the reference ligand, and the approach used to compare the interactions formed by the docked ligand with those formed by the reference ligand in the crystallographic structure. Here, we propose a new method based on the conformational dynamics of a single protein-ligand reference complex to improve the biased selection of ligands with specific pharmacological properties in a structure-based virtual screening exercise. Interactions patterns between a reference agonist and the receptor, here exemplified on the β2 adrenergic receptor, were extracted from molecular dynamics simulations of the agonist/receptor complex and encoded in graphs used to train a one-class machine learning classifier. Different conditions were tested: low to high affinity agonists, varying simulation duration, considering or ignoring hydrophobic contacts, and tuning of the classifier parametrization. The best models applied to post-process raw data from retrospective virtual screening obtained by docking of test libraries effectively filtered out irrelevant poses, discarding inactive and non-agonist ligands while identifying agonists. Taken together, our results suggest that consistency of the binding mode during the simulation is a key to the success of the method.

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Agonist binding by the β2-adrenergic receptor: an effect of receptor conformation on ligand association-dissociation characteristics.

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