Exploring a new ligand binding site of G protein-coupled receptors

AbstractElucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein–ligand binding, especially the drug recognition of GPCRs.

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Ancestral reconstruction of the ligand-binding pocket of Family C G protein-coupled receptors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0604717103 ◽

2006 ◽

Vol 103 (38) ◽

pp. 14050-14055 ◽

Cited By ~ 31

Author(s):

D. Kuang ◽

Y. Yao ◽

D. MacLean ◽

M. Wang ◽

D. R. Hampson ◽

...

Keyword(s):

Ligand Binding ◽

G Protein ◽

Binding Pocket ◽

G Protein Coupled Receptors ◽

Ancestral Reconstruction ◽

Ligand Binding Pocket ◽

G Protein Coupled

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Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand binding site

FEBS Letters ◽

10.1016/j.febslet.2008.08.022 ◽

2008 ◽

Vol 582 (23-24) ◽

pp. 3335-3342 ◽

Cited By ~ 27

Author(s):

Steffen Wolf ◽

Marcus Böckmann ◽

Udo Höweler ◽

Jürgen Schlitter ◽

Klaus Gerwert

Keyword(s):

Ligand Binding ◽

Binding Site ◽

G Protein ◽

Homology Model ◽

Ligand Binding Site ◽

Dynamic Features ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Structural Requirements for the Activation of the Human Growth Hormone Secretagogue Receptor by Peptide and Nonpeptide Secretagogues

Molecular Endocrinology ◽

10.1210/mend.12.1.0051 ◽

1998 ◽

Vol 12 (1) ◽

pp. 137-145 ◽

Cited By ~ 44

Author(s):

Scott D. Feighner ◽

Andrew D. Howard ◽

Kristine Prendergast ◽

Oksana C. Palyha ◽

Donna L. Hreniuk ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

G Protein ◽

Human Growth ◽

G Protein Coupled Receptors ◽

Site Directed Mutagenesis ◽

Agonist Binding ◽

Growth Hormone Secretagogue Receptor ◽

Growth Hormone Secretagogue ◽

G Protein Coupled

Abstract Antibodies raised against an intracellular and extracellular domain of the GH secretagogue receptor (GHS-R) confirmed that its topological orientation in the lipid bilayer is as predicted for G protein-coupled receptors with seven transmembrane domains. A strategy for mapping the agonist-binding site of the human GHS-R was conceived based on our understanding of ligand binding in biogenic amine and peptide hormone G protein-coupled receptors. Using site-directed mutagenesis and molecular modeling, we classified GHS peptide and nonpeptide agonist binding in the context of its receptor environment. All peptide and nonpeptide ligand classes shared a common binding domain in transmembrane (TM) region 3 of the GHS-R. This finding was based on TM-3 mutation E124Q, which eliminated the counter-ion to the shared basic N+ group of all GHSs and resulted in a nonfunctional receptor. Restoration of function for the E124Q mutant was achieved by a complementary change in the MK-0677 ligand through modification of its amine side-chain to the corresponding alcohol. Contacts in other TM domains [TM-2 (D99N), TM-5 (M213K, S117A), TM-6 (H280F), and extracellular loop 1 (C116A)] of the receptor revealed specificity for the different peptide, benzolactam, and spiroindolane GHSs. GHS-R agonism, therefore, does not require identical disposition of all agonist classes at the ligand-binding site. Our results support the hypothesis that the ligand-binding pocket in the GHS-R is spatially disposed similarly to the well characterized catechol-binding site in theβ 2-adrenergic receptor.

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A minimal ligand binding pocket within a network of correlated mutations identified by multiple sequence and structural analysis of G protein coupled receptors

BMC Biophysics ◽

10.1186/2046-1682-5-13 ◽

2012 ◽

Vol 5 (1) ◽

pp. 13 ◽

Cited By ~ 5

Author(s):

Subhodeep Moitra ◽

Kalyan C Tirupula ◽

Judith Klein-Seetharaman ◽

Christopher Langmead

Keyword(s):

Structural Analysis ◽

Ligand Binding ◽

G Protein ◽

Binding Pocket ◽

G Protein Coupled Receptors ◽

Multiple Sequence ◽

Ligand Binding Pocket ◽

Correlated Mutations ◽

G Protein Coupled

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Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800756115 ◽

2018 ◽

Vol 115 (12) ◽

pp. 3036-3041 ◽

Cited By ~ 43

Author(s):

Yinglong Miao ◽

J. Andrew McCammon

Keyword(s):

Protein Binding ◽

G Protein ◽

Protein Interactions ◽

Intracellular Signaling ◽

Md Simulations ◽

Binding Pocket ◽

Signaling Proteins ◽

Protein Protein Interactions ◽

Intracellular Loops ◽

G Protein Coupled

Protein–protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein–protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR–nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein–protein interactions.

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A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity

Science Signaling ◽

10.1126/scisignal.aaw5885 ◽

2020 ◽

Vol 13 (617) ◽

pp. eaaw5885 ◽

Cited By ~ 5

Author(s):

Marta Sanchez-Soto ◽

Ravi Kumar Verma ◽

Blair K. A. Willette ◽

Elizabeth C. Gonye ◽

Annah M. Moore ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

G Protein ◽

Structural Basis ◽

Protein Signaling ◽

Ligand Binding Site ◽

Intracellular Loop ◽

Gpcr Signaling ◽

Biased Signaling ◽

Dynamics Simulations

Signaling bias is the propensity for some agonists to preferentially stimulate G protein–coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein–biased agonist of the D2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β2-adrenergic receptor (β2R) to build β2R-WT and β2R-Y1995.38A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y1995.38A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.

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