Towards A Molecular Understanding of The Cannabinoid Related Orphan Receptor GPR18: A Focus on Its Constitutive Activity

The orphan G-protein coupled receptor (GPCR), GPR18, has been recently proposed as a potential member of the cannabinoid family as it recognizes several endogenous, phytogenic, and synthetic cannabinoids. Potential therapeutic applications for GPR18 include intraocular pressure, metabolic disorders, and cancer. GPR18 has been reported to have high constitutive activity, i.e., activation/signaling occurs in the absence of an agonist. This activity can be reduced significantly by the A3.39N mutation. At the intracellular (IC) ends of (transmembrane helices) TMH3 and TMH6 in GPCRs, typically, a pair of oppositely charged amino acids form a salt bridge called the “ionic lock”. Breaking of this salt bridge creates an IC opening for coupling with G protein. The GPR18 “ionic lock” residues (R3.50/S6.33) can form only a hydrogen bond. In this paper, we test the hypothesis that the high constitutive activity of GPR18 is due to the weakness of its “ionic lock” and that the A3.39N mutation strengthens this lock. To this end, we report molecular dynamics simulations of wild-type (WT) GPR18 and the A3.39N mutant in fully hydrated (POPC) phophatidylcholine lipid bilayers. Results suggest that in the A3.39N mutant, TMH6 rotates and brings R3.50 and S6.33 closer together, thus strengthening the GPR18 “ionic lock”.

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Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites

10.1101/2020.10.22.349522 ◽

2020 ◽

Author(s):

Valeria Marquez-Miranda ◽

Maximiliano Rojas ◽

Yorley Duarte ◽

Ignacio Diaz-Franulic ◽

Miguel Holmgren ◽

...

Keyword(s):

Transmembrane Domain ◽

Lung Epithelial Cells ◽

Transmembrane Helices ◽

Cellular Processes ◽

Charged Amino Acids ◽

Coordination Sites ◽

Lung Epithelial ◽

Dynamics Simulations ◽

Positively Charged

AbstractSARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimer’s cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domain’s potential permeation pathway has six constrictions narrowing to about 1 Å in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavity’s electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.

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Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase

Biochemical Journal ◽

10.1042/bj3090837 ◽

1995 ◽

Vol 309 (3) ◽

pp. 837-843 ◽

Cited By ~ 81

Author(s):

D Eggerickx ◽

J F Denef ◽

O Labbe ◽

Y Hayashi ◽

S Refetoff ◽

...

Keyword(s):

Adenylate Cyclase ◽

G Protein ◽

Rnase Protection Assay ◽

Biological Significance ◽

Orphan Receptor ◽

Constitutive Activity ◽

G Protein Coupled Receptor ◽

Rnase Protection ◽

G Protein Coupled ◽

Stimulation Of

A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed.

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Charged Residues at the Intracellular Boundary of Transmembrane Helices 2 and 3 Independently Affect Constitutive Activity of Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m007885200 ◽

2000 ◽

Vol 276 (2) ◽

pp. 1376-1382 ◽

Cited By ~ 31

Author(s):

Hao H. Ho ◽

Nimalja Ganeshalingam ◽

Avia Rosenhouse-Dantsker ◽

Roman Osman ◽

Marvin C. Gershengorn

Keyword(s):

G Protein ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Constitutive Activity ◽

Transmembrane Helices ◽

G Protein Coupled Receptor ◽

Charged Residues ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

G Protein Coupled

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Engineering Salt Bridge Networks between Transmembrane Helices Confers Thermostability in G-Protein-Coupled Receptors

Journal of Chemical Theory and Computation ◽

10.1021/acs.jctc.8b00602 ◽

2018 ◽

Vol 14 (12) ◽

pp. 6574-6585 ◽

Cited By ~ 6

Author(s):

Soumadwip Ghosh ◽

Tobias Bierig ◽

Sangbae Lee ◽

Suvamay Jana ◽

Adelheid Löhle ◽

...

Keyword(s):

G Protein ◽

Salt Bridge ◽

G Protein Coupled Receptors ◽

Transmembrane Helices ◽

Bridge Networks ◽

G Protein Coupled

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Detection of Side Chain Rearrangements Mediating the Motions of Transmembrane Helices in Molecular Dynamics Simulations of G Protein-Coupled Receptors

Computational and Structural Biotechnology Journal ◽

10.1016/j.csbj.2017.01.001 ◽

2017 ◽

Vol 15 ◽

pp. 131-137 ◽

Cited By ~ 7

Author(s):

Zied Gaieb ◽

Dimitrios Morikis

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

G Protein ◽

G Protein Coupled Receptors ◽

Side Chain ◽

Transmembrane Helices ◽

Dynamics Simulations ◽

G Protein Coupled

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Structures of the Otopetrin Proton Channels Otop1 and Otop3

10.1101/542308 ◽

2019 ◽

Author(s):

Kei Saotome ◽

Bochuan Teng ◽

Che Chun (Alex) Tsui ◽

Wen-Hsin Lee ◽

Yu-Hsiang Tu ◽

...

Keyword(s):

Taste Receptor ◽

Salt Bridge ◽

Proton Channel ◽

Structural Basis ◽

Transmembrane Helices ◽

Proton Channels ◽

Selective Channel ◽

Functional Relevance ◽

Dynamics Simulations ◽

Lipid Nanodiscs

Otopetrins (Otop1-Otop3) comprise one of only two known eukaryotic proton-selective channel families. Otop1 is required for formation of otoconia and is a candidate mammalian sour taste receptor. Here, we report cryo-EM structures of zebrafish Otop1 and chicken Otop3 in lipid nanodiscs. The structures reveal a dimeric architecture of Otopetrins with each subunit consisting of twelve transmembrane helices divided into structurally related N and C domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a cavernous central tunnel. Two hydrophilic vestibules, as well as the intrasubunit interface between N and C domains, form conduits for water entry into the membrane plane in molecular dynamics simulations, suggesting they each could provide pathways for proton conduction. We also demonstrate the functional relevance of a salt bridge in the C domain vestibule by mutagenesis. Our results provide a structural basis for understanding the function of the Otopetrin proton channel family.

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A decoy heterotrimeric Gα protein has substantially reduced nucleotide binding but retains nucleotide-independent interactions with its cognate RGS protein and Gβγ dimer

10.1101/795088 ◽

2019 ◽

Author(s):

Fei Lou ◽

Tigran M. Abramyan ◽

Haiyan Jia ◽

Alexander Tropsha ◽

Alan M. Jones

Keyword(s):

G Protein ◽

Protein Interactions ◽

Salt Bridge ◽

Nucleotide Binding ◽

Bound State ◽

Dominant Negative ◽

Guanine Nucleotide ◽

Rgs Domain ◽

Dynamics Simulations

ABSTRACTPlants uniquely have a family of proteins called extra-large G proteins (XLG) that share homology in their C-terminal half with the canonical Gα subunits; we carefully detail here that Arabidopsis XLG2 lacks critical residues requisite for nucleotide binding and hydrolysis which is consistent with our quantitative analyses. Based on microscale thermophoresis, Arabidopsis XLG2 binds GTPγS with an affinity 100-1000 times lower than that to canonical Gα subunits. This means that given the concentration range of guanine nucleotide in plant cells, XLG2 is not likely bound by GTP in vivo. Homology modeling and molecular dynamics simulations provide a plausible mechanism for the poor nucleotide binding affinity of XLG2. Simulations indicate substantially stronger salt bridge networks formed by several key amino-acid residues of AtGPA1 which are either misplaced or missing in XLG2. These residues in AtGPA1 not only maintain the overall shape and integrity of the apoprotein cavity but also increase the frequency of favorable nucleotide-protein interactions in the nucleotide-bound state. Despite this loss of nucleotide dependency, XLG2 binds the RGS domain of AtRGS1 with an affinity similar to the Arabidopsis AtGPA1 in its apo-state and about 2 times lower than AtGPA1 in its transition state. In addition, XLG2 binds the Gβγ dimer with an affinity similar to that of AtGPA1. XLG2 likely acts as a dominant negative Gα protein to block G protein signaling. We propose that XLG2, independent of guanine nucleotide binding, regulates the active state of the canonical G protein pathway directly by sequestering Gβγ and indirectly by promoting heterodimer formation.

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Repulsive Separation of the Cytoplasmic Ends of Transmembrane Helices 3 and 6 Is Linked to Receptor Activation in a Novel Thyrotropin Receptor Mutant (M626I)

Molecular Endocrinology ◽

10.1210/me.2005-0339 ◽

2006 ◽

Vol 20 (4) ◽

pp. 893-903 ◽

Cited By ~ 24

Author(s):

Usanee Ringkananont ◽

Joost Van Durme ◽

Lucia Montanelli ◽

Figen Ugrasbul ◽

Y. Miles Yu ◽

...

Keyword(s):

G Protein ◽

Surface Expression ◽

Receptor Activation ◽

Cell Surface Expression ◽

Constitutive Activity ◽

Transmembrane Helices ◽

Wild Type ◽

Gpcr Activation ◽

Camp Generation ◽

Site Directed Spin Labeling

Abstract Ligand-dependent activation of G protein-coupled receptors (GPCRs) involves repositioning of the juxtacytoplasmic ends of transmembrane helices TM3 and TM6. This concept, inferred from site-directed spin labeling studies, is supported by chemical cross-linking of the cytoplasmic ends of TM3 and TM6 blocking GPCR activation. Here we report a novel constitutive active mutation (M626I) in TM6 of the TSH receptor (TSHR), identified in affected members of a family with nonautoimmune hyperthyroidism. The specific constitutive activity of M626I, measured by its basal cAMP generation corrected for cell surface expression, was 13-fold higher than that of wild-type TSHR. Homology modeling of the TSHR serpentine domain based on the rhodopsin crystal structure suggests that M626 faces the side chain of I515 of TM3 near the membrane-cytoplasmic junction. Steric hindrance of the introduced isoleucine by I515 is consistent with the fact that shorter or more flexible side chains at position 626 did not increase constitutivity. Furthermore, a reciprocal mutation at position 515 (I515M), when introduced into the M626I background, acts as revertant mutation by allowing accommodation of the isoleucine sidechain at position 626 and fully restoring the constitutive activity to the level of wild-type TSHR. Thus, repulsive separation of the juxtacytoplasmic TM6 and TM3 in the M626I model conclusively demonstrates a direct link between the opening of this cytoplasmic face of the receptor structure and G protein coupling.

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Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis, in lipid bilayers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1415908112 ◽

2014 ◽

Vol 112 (2) ◽

pp. E119-E126 ◽

Cited By ~ 37

Author(s):

Nabanita Das ◽

Jian Dai ◽

Ivan Hung ◽

Malini Rajagopalan ◽

Huan-Xiang Zhou ◽

...

Keyword(s):

Molecular Dynamics ◽

Mycobacterium Tuberculosis ◽

Lipid Bilayers ◽

Transmembrane Domain ◽

Magic Angle Spinning ◽

Magic Angle ◽

Transmembrane Helices ◽

Oriented Sample ◽

Angle Spinning ◽

Dynamics Simulations

The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid–crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA’s partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines.

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