scholarly journals Nucleic acid ligands act as a PAM and agonist depending on the intrinsic ligand binding state of P2RY2

2021 ◽  
Vol 118 (18) ◽  
pp. e2019497118
Author(s):  
Masaki Takahashi ◽  
Ryo Amano ◽  
Michiru Ozawa ◽  
Anna Martinez ◽  
Kazumasa Akita ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Ligand Binding ◽  
Purinergic Receptor ◽  
Partial Agonist ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Gpcr Activation ◽  
Virus Like Particle ◽  
A Cell ◽  
Biochemical Analyses

G protein–coupled receptors (GPCRs) play diverse roles in physiological processes, and hence the ligands to modulate GPCRs have served as important molecules in biological and pharmacological approaches. However, the exploration of novel ligands for GPCR still remains an arduous challenge. In this study, we report a method for the discovery of nucleic acid ligands against GPCRs by an advanced RNA aptamer screening technology that employs a virus-like particle (VLP), exposing the GPCR of interest. An array of biochemical analyses coupled with a cell-based assay revealed that one of the aptamers raised against purinergic receptor P2Y2 (P2RY2), a GPCR, exhibits an activation potency to unliganded receptor and prohibits a further receptor activation by endogenous ligand, behaving like a partial agonist. However, the aptamer enhances the activity of intrinsic ligand-binding P2RY2, thereby acting as a positive allosteric modulator (PAM) to liganded receptor. Our findings demonstrate that the nucleic acid aptamer conditionally exerts PAM and agonist effects on GPCRs, depending on their intrinsic ligand binding state. These results indicate the validity of our VLP-based aptamer screening targeting GPCR and reemphasize the great potential of nucleic acid ligands for exploring the GPCR activation mechanism and therapeutic applications.

scholarly journals Universal activation mechanism of class A GPCRs

2019 ◽  
Author(s):  
Qingtong Zhou ◽  
Dehua Yang ◽  
Meng Wu ◽  
Yu Guo ◽  
Wangjing Guo ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Coupling Region ◽  
Activation Pathway ◽  
Class A ◽  
Gpcr Activation

AbstractClass A G protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. GPCR activation is an allosteric process that links agonist binding to G protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue-level changes of this trigger remain less well understood. Here, by analyzing over 230 high-resolution structures of class A GPCRs, we discovered a modular, universal GPCR activation pathway that unites previous findings into a common activation mechanism, directly linking the bottom of ligand-binding pocket with G protein-coupling region. We suggest that the modular nature of the universal GPCR activation pathway allowed for the decoupling of the evolution of the ligand binding site, G protein binding region and the residues important for receptor activation. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.

scholarly journals Common activation mechanism of class A GPCRs

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Qingtong Zhou ◽  
Dehua Yang ◽  
Meng Wu ◽  
Yu Guo ◽  
Wanjing Guo ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Conformational Changes ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Activation Mechanism ◽  
Activation Pathway ◽  
Class A ◽  
Gpcr Activation ◽  
The Common

Class A G-protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. Understanding receptor activation mechanism is critical for discovering novel therapeutics since about one-third of all marketed drugs target members of this family. GPCR activation is an allosteric process that couples agonist binding to G-protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue level changes of this movement remain less well understood. Here, we report a framework to quantify conformational changes. By analyzing the conformational changes in 234 structures from 45 class A GPCRs, we discovered a common GPCR activation pathway comprising of 34 residue pairs and 35 residues. The pathway unifies previous findings into a common activation mechanism and strings together the scattered key motifs such as CWxP, DRY, Na+ pocket, NPxxY and PIF, thereby directly linking the bottom of ligand-binding pocket with G-protein coupling region. Site-directed mutagenesis experiments support this proposition and reveal that rational mutations of residues in this pathway can be used to obtain receptors that are constitutively active or inactive. The common activation pathway provides the mechanistic interpretation of constitutively activating, inactivating and disease mutations. As a module responsible for activation, the common pathway allows for decoupling of the evolution of the ligand binding site and G-protein-binding region. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.

scholarly journals Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Max Bernhard ◽  
Bodo Laube
Keyword(s):  
Ligand Binding ◽  
Partial Agonist ◽  
Glycine Receptor ◽  
Three Dimensional ◽  
Receptor Activation ◽  
Cell System ◽  
Activation Mechanism ◽  
Hek293 Cells ◽  
Dimensional Structure ◽  
Conformational States

Abstract The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel family (pLGIC), displays remarkable variations in the affinity and efficacy of the full agonist glycine and the partial agonist taurine depending on the cell system used. Despite detailed insights in the GlyR three-dimensional structure and activation mechanism, little is known about conformational rearrangements induced by these agonists. Here, we characterized the conformational states of the α1 GlyR upon binding of glycine and taurine by microscale thermophoresis expressed in HEK293 cells and Xenopus oocytes after solubilization in amphipathic styrene-maleic acid copolymer nanodiscs. Our results show that glycine and taurine induce different conformational transitions of the GlyR upon ligand binding. In contrast, the variability of agonist affinity is not mediated by an altered conformational change. Thus, our data shed light on specific agonist induced conformational features and mechanisms of pLGIC upon ligand binding determining receptor activation in native environments.

scholarly journals Receptor-wide Determinants of G Protein Coupling Selectivity in Aminergic GPCRs

2021 ◽  
Author(s):  
Berkay Selçuk ◽  
Ismail Erol ◽  
Serdar Durdağı ◽  
Ogun Adebali
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Md Simulations ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Selective Activation ◽  
G Protein Coupling ◽  
Protein Coupling ◽  
Gpcr Activation ◽  
Activation Mechanisms

AbstractG protein-coupled receptors (GPCRs) induce signal transduction pathways through coupling to four main subtypes of G proteins (Gs, Gi, Gq, G12/13), selectively. However, G protein selective activation mechanisms and residual determinants in GPCRs have remained obscure. Here, we identified conserved G protein selective activation mechanisms determining receptors’ ability to couple to a type of G protein. Herein, we performed an extensive phylogenetic analysis and identified specifically conserved residues for the receptors having similar coupling profiles in each aminergic receptor. By integrating our methodology of differential evolutionary conservation of G protein-specific amino acids with structural analyses, we identified selective activation networks for Gs, Gi1, Go, and Gq. We found that G protein selectivity is determined by not only the G protein interaction site but also other parts of the receptor including the ligand binding pocket. To validate our findings, we further studied an amino acid residue that we revealed as a selectivity-determining in Gs coupling and performed molecular dynamics (MD) simulations. We showed that previously uncharacterized Glycine at position 7×41 plays an important role in both receptor activation and Gs coupling. Finally, we gathered our results into a comprehensive model of G protein selectivity called “sequential switches of activation” describing three main molecular switches controlling GPCR activation: ligand binding, G protein selective activation mechanisms and G protein contact. We believe that our work provides a broader view on receptor-level determinants of G protein coupling selectivity.

scholarly journals Mechanistic basis for the activation of plant membrane receptor kinases by SERK-family coreceptors

2018 ◽  
Vol 115 (13) ◽  
pp. 3488-3493 ◽  
Author(s):  
Ulrich Hohmann ◽  
Julia Santiago ◽  
Joël Nicolet ◽  
Vilde Olsson ◽  
Fabio M. Spiga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Specific Binding ◽  
Peptide Hormone ◽  
Receptor Activation ◽  
Kinase Domain ◽  
Membrane Receptor ◽  
Activation Mechanism ◽  
In Planta ◽  
Affinity Ligand ◽  
Receptor Kinases

Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor–coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor–coreceptor heteromerization in planta. A functional BRI1–HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development.

scholarly journals Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists

2005 ◽  
Vol 280 (23) ◽  
pp. 22165-22171 ◽  
Author(s):  
Gayathri Swaminath ◽  
Xavier Deupi ◽  
Tae Weon Lee ◽  
Wen Zhu ◽  
Foon Sun Thian ◽  
...  
Keyword(s):  
Binding Site ◽  
Aromatic Ring ◽  
Partial Agonist ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Molecular Probe ◽  
Toggle Switch ◽  
Partial Agonists ◽  
Gpcr Activation ◽  
Multistep Process

The β2 adrenergic receptor (β2AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The β2AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between β2AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the β2AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.

scholarly journals Correlated motions of conserved polar motifs lay out a plausible mechanism of G protein-coupled receptor activation.

2020 ◽  
Author(s):  
Argha Mitra ◽  
Arijit Sarkar ◽  
Marton Richard Szabo ◽  
Attila Borics
Keyword(s):  
G Protein ◽  
Intracellular Signaling ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Current Theory ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Binding Interface ◽  
Gpcr Activation ◽  
G Protein Coupled

Recent advancements in the field of experimental structural biology have provided high-resolution structures of active and inactive state G protein-coupled receptors (GPCRs), a highly important pharmaceutical target family, but the process of transition between these states is poorly understood. According to the current theory, GPCRs exist in structurally distinct, dynamically interconverting functional states of which populations are shifted upon binding of ligands and intracellular signaling proteins. However, explanation of the activation mechanism on an entirely structural basis gets complicated when multiple activation pathways and active receptor states are considered. Our unbiased, atomistic molecular dynamics simulations of the mu-opioid receptor in a physiological environment revealed that external stimulus is propagated to the intracellular surface of the receptor through subtle, concerted movements of highly conserved polar amino acid side chains along the 7th transmembrane helix. To amend the widely accepted theory we suggest that the initiation event of GPCR activation is the shift of macroscopic polarization between the ortho- and allosteric binding pockets and the intracellular G protein-binding interface.

scholarly journals Allosteric effect of nanobody binding on ligand-specific active states of the β2-Adrenergic Receptor

2021 ◽  
Author(s):  
Yue Chen ◽  
Oliver Fleetwood ◽  
Sergio Perez-Conesa ◽  
Lucie Delemotte
Keyword(s):  
G Protein ◽  
Conformational Changes ◽  
Adrenergic Receptor ◽  
Partial Agonist ◽  
Transmembrane Helix ◽  
Active State ◽  
Supervised Machine Learning ◽  
Activation Mechanism ◽  
Gpcr Activation ◽  
Loop 2

Nanobody binding stabilizes the active state of G-protein-coupled receptor (GPCR) and modulates its 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 beta2 adrenergic receptor (beta2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes a highly active state of the beta2AR with a ~14 A outward movement of transmembrane helix 6 and close proximity of transmembrane (TM) helices 5 and 7. This is further 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 intercellular 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 can enhance the communication between the binding sites of Nb80 and of the ligand. We identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in presence of full agonist and G-protein biased partial agonist, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which could be useful for structure-based drug discovery.

scholarly journals Correlated Motions of Conserved Polar Motifs Lay out a Plausible Mechanism of G Protein-Coupled Receptor Activation

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 670
Author(s):  
Argha Mitra ◽  
Arijit Sarkar ◽  
Márton Richárd Szabó ◽  
Attila Borics
Keyword(s):  
G Protein ◽  
Intracellular Signaling ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Binding Interface ◽  
Gpcr Activation ◽  
Macroscopic Polarization ◽  
G Protein Coupled

Recent advancements in the field of experimental structural biology have provided high-resolution structures of active and inactive state G protein-coupled receptors (GPCRs), a highly important pharmaceutical target family, but the process of transition between these states is poorly understood. According to the current theory, GPCRs exist in structurally distinct, dynamically interconverting functional states of which populations are shifted upon binding of ligands and intracellular signaling proteins. However, explanation of the activation mechanism, on an entirely structural basis, gets complicated when multiple activation pathways and active receptor states are considered. Our unbiased, atomistic molecular dynamics simulations of the μ opioid receptor (MOP) revealed that transmission of external stimulus to the intracellular surface of the receptor is accompanied by subtle, concerted movements of highly conserved polar amino acid side chains along the 7th transmembrane helix. This may entail the rearrangement of polar species and the shift of macroscopic polarization in the transmembrane domain, triggered by agonist binding. Based on our observations and numerous independent indications, we suggest amending the widely accepted theory that the initiation event of GPCR activation is the shift of macroscopic polarization between the ortho- and allosteric binding pockets and the intracellular G protein-binding interface.

Using FRET to Measure the Time it Takes for a Cell to Destroy a Virus

2019 ◽  
Author(s):  
Candace E. Benjamin ◽  
Zhuo Chen ◽  
Olivia Brohlin ◽  
Hamilton Lee ◽  
Stefanie Boyd ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Rhodamine B ◽  
Virus Like Particle ◽  
A Cell ◽  
Viral Nanoparticles ◽  
The Stability ◽  
Open Question ◽  
Viral Surface ◽  
Fret Pair

<div><div><div><p>The emergence of viral nanotechnology over the preceding two decades has created a number of intellectually captivating possible translational applications; however, the in vitro fate of the viral nanoparticles in cells remains an open question. Herein, we investigate the stability and lifetime of virus-like particle (VLP) Qβ - a representative and popular VLP for several applications - following cellular uptake. By exploiting the available functional handles on the viral surface, we have orthogonally installed the known FRET pair, FITC and Rhodamine B, to gain insight of the particle’s behavior in vitro. Based on these data, we believe VLPs undergo aggregation in addition to the anticipated proteolysis within a few hours of cellular uptake.</p></div></div></div>

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