scholarly journals Allosteric ligands control the activation of a class C GPCR heterodimer by acting at the transmembrane interface

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lei Liu ◽  
Zhiran Fan ◽  
Xavier Rovira ◽  
Li Xue ◽  
Salomé Roux ◽  
...  
Keyword(s):  
Binding Site ◽  
Drug Targets ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Site Directed Mutagenesis ◽  
Fine Tuning ◽  
Ion Binding ◽  
Allosteric Modulators ◽  
Class A ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and to validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAM tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface.

scholarly journals Intracellular passage of Na+ in an active state g-protein coupled receptor

2017 ◽  
Author(s):  
Owen N. Vickery ◽  
Catarina A. Carvalheda ◽  
Saheem A. Zaidi ◽  
Andrei V. Pisliakov ◽  
Vsevolod Katritch ◽  
...  
Keyword(s):  
Binding Site ◽  
Drug Targets ◽  
Transmembrane Domain ◽  
Cell Signalling ◽  
Effector Proteins ◽  
Active State ◽  
Gpcr Activation ◽  
Activated State ◽  
Voltage Gradients ◽  
G Protein Coupled

ABSTRACTPlaying a central role in cell signalling, GPCRs have evolved into the largest superfamily of membrane proteins and form the majority of drug targets in humans. How extracellular agonist binding triggers the activation of GPCRs and associated intracellular effector proteins remains, however, poorly understood. High resolution structural studies have recently revealed that inactive class-A GPCRs harbour a conserved binding site for Na+ ions in the centre of their transmembrane domain, accessible from the extracellular space. Here, we show that the opening of a conserved hydrated channel in the activated state receptors allows the Na+ ion to egress from its binding site into the cytosol. Coupled with protonation changes, this ion movement occurs without significant energy barriers, and can be driven by physiological transmembrane ion and voltage gradients. We propose that Na+ ion exchange with the cytosol is a key step in GPCR activation, which locks receptors in long-lived active-state conformations.

scholarly journals Molecular determinants underlying DS2 activity at δ-containing GABAA receptors

2021 ◽  
Author(s):  
Christina B. Falk-Petersen ◽  
Frederik Rostrup ◽  
Rebekka Löffler ◽  
Stine Buchleithner ◽  
Kasper Harpsøe ◽  
...  
Keyword(s):  
Binding Site ◽  
Drug Targets ◽  
Transmembrane Domain ◽  
Direct Interaction ◽  
Binding Pocket ◽  
Docking Studies ◽  
Tonic Inhibition ◽  
Site Directed Mutagenesis ◽  
Receptor Subtypes ◽  
Molecular Determinants

AbstractDelta selective compound 2 (DS2) is one of the most widely used tools to study selective actions mediated by δ subunit-containing GABAA receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive.Using a combination of computational modeling, site-directed mutagenesis and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α4β1δ receptors: an α4(+)δ(-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αβγ2 receptors, and two sites in the transmembrane domain (TMD); one in the α4(+)β1(-) and one in the α4(-)β1(+) interface, with the α4(-)β1(+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α4(+)β1(-) interface, either α4(S303L) of the α4(+)-side or β1(I289Q) of the β1(-)-side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patchclamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points towards a functional selectivity of DS2 and its analogs via a surprisingly well-conserved binding pocket in the TMD.Significance statementδ-Containing GABAA receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition. Yet, no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the know compound DS2, the ground is laid for design of ligands that selectively target δ-containing GABAA receptor subtypes, for better understanding of tonic inhibition, and, ultimately, for rational development of novel drugs.

Universality of the Sodium Ion Binding Mechanism in Class A G-Protein-Coupled Receptors

2018 ◽  
Vol 130 (12) ◽  
pp. 3102-3107 ◽  
Author(s):  
Balaji Selvam ◽  
Zahra Shamsi ◽  
Diwakar Shukla
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Sodium Ion ◽  
Ion Binding ◽  
Binding Mechanism ◽  
Class A ◽  
G Protein Coupled

scholarly journals A two-domain elevator mechanism for sodium/proton antiport

2014 ◽  
Vol 70 (a1) ◽  
pp. C1048-C1048
Author(s):  
Chiara Lee ◽  
Hae Joo Kang ◽  
Christoph von Ballmoos ◽  
Simon Newstead ◽  
Povilas Uzdavinys ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Drug Targets ◽  
Thermus Thermophilus ◽  
Ion Binding ◽  
Dimerization Domain ◽  
Core Domain ◽  
Large Rotation ◽  
The Core ◽  
Dynamics Simulations

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 200against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

scholarly journals Fine Tuning Muscarinic Acetylcholine Receptor Signaling Through Allostery and Bias

2021 ◽  
Vol 11 ◽  
Author(s):  
Emma T. van der Westhuizen ◽  
K. H. Christopher Choy ◽  
Celine Valant ◽  
Simon McKenzie-Nickson ◽  
Sophie J. Bradley ◽  
...  
Keyword(s):  
Binding Site ◽  
Muscarinic Acetylcholine Receptors ◽  
Fine Tuning ◽  
Drug Candidate ◽  
Receptor Subtype ◽  
Allosteric Modulators ◽  
Biased Agonism ◽  
Muscarinic Acetylcholine ◽  
Subtype Selectivity ◽  
Subtype Selective

The M1 and M4 muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, in particular for Alzheimer’s disease and schizophrenia. Due to high sequence homology, selective targeting of any of the M1-M5 mAChRs through the endogenous ligand binding site has been notoriously difficult to achieve. With the discovery of highly subtype selective mAChR positive allosteric modulators in the new millennium, selectivity through targeting an allosteric binding site has opened new avenues for drug discovery programs. However, some hurdles remain to be overcome for these promising new drug candidates to progress into the clinic. One challenge is the potential for on-target side effects, such as for the M1 mAChR where over-activation of the receptor by orthosteric or allosteric ligands can be detrimental. Therefore, in addition to receptor subtype selectivity, a drug candidate may need to exhibit a biased signaling profile to avoid such on-target adverse effects. Indeed, recent studies in mice suggest that allosteric modulators for the M1 mAChR that bias signaling toward specific pathways may be therapeutically important. This review brings together details on the signaling pathways activated by the M1 and M4 mAChRs, evidence of biased agonism at these receptors, and highlights pathways that may be important for developing new subtype selective allosteric ligands to achieve therapeutic benefit.

scholarly journals A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

2004 ◽  
Vol 24 (5) ◽  
pp. 2041-2051 ◽  
Author(s):  
Jennifer C. Lin ◽  
Ken Duell ◽  
James B. Konopka
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Transmembrane Domains ◽  
Consecutive Series ◽  
Subsequent Step ◽  
G Protein Coupled ◽  
Specific Reagent

ABSTRACT The α-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

scholarly journals Discovery of Novel Allosteric Modulators Targeting an Extra-Helical Binding Site of GLP-1R Using Structure- and Ligand-Based Virtual Screening

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 929
Author(s):  
Qingtong Zhou ◽  
Wanjing Guo ◽  
Antao Dai ◽  
Xiaoqing Cai ◽  
Márton Vass ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Binding Site ◽  
Computational Approach ◽  
G Protein Coupled Receptors ◽  
Screening Methods ◽  
Allosteric Modulators ◽  
Downstream Signaling ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
G Protein Coupled

Allosteric modulators have emerged with many potential pharmacological advantages as they do not compete the binding of agonist or antagonist to the orthosteric sites but ultimately affect downstream signaling. To identify allosteric modulators targeting an extra-helical binding site of the glucagon-like peptide-1 receptor (GLP-1R) within the membrane environment, the following two computational approaches were applied: structure-based virtual screening with consideration of lipid contacts and ligand-based virtual screening with the maintenance of specific allosteric pocket residue interactions. Verified by radiolabeled ligand binding and cAMP accumulation experiments, two negative allosteric modulators and seven positive allosteric modulators were discovered using structure-based and ligand-based virtual screening methods, respectively. The computational approach presented here could possibly be used to discover allosteric modulators of other G protein-coupled receptors.

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