scholarly journals Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

2020 ◽  
Author(s):  
Gerhard Wagner ◽  
Meng Zhang ◽  
Miao Gui ◽  
Zi-Fu Wang ◽  
Christoph Gorgulla ◽  
...  
Keyword(s):  
Complex Formation ◽  
G Protein ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Protein Complex ◽  
Lipid Membrane ◽  
Protein Complexes ◽  
Structural Basis ◽  
Downstream Signaling

Abstract G protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, structural studies of the complex in a physiological lipid membrane environment are lacking. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gai1b1g1 protein in two conformational states, resolved to 4.1 and 4.2 Å resolution. The structures were determined in lipid bilayer without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

scholarly journals Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

2020 ◽  
Author(s):  
Meng Zhang ◽  
Miao Gui ◽  
Zi-Fu Wang ◽  
Christoph Gorgulla ◽  
James J Yu ◽  
...  
Keyword(s):  
Complex Formation ◽  
G Protein ◽  
G Proteins ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Protein Complex ◽  
Lipid Membrane ◽  
Complex Structure ◽  
Underlying Mechanism ◽  
Structural Basis

AbstractG protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals1,2. Although several structures have been solved for GPCR-G protein complexes3–17, structural studies of the complex in a physiological lipid membrane environment are lacking. Additionally, most previous studies required additional antibodies/nanobodies and/or engineered G proteins for complex stabilization. In the absence of a native complex structure, the underlying mechanism of G protein activation leading to GDP/GTP exchange remains unclear. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gαi1β1γ1 protein in two conformational states, resolved to 4.1 and 4.2 Å resolution. The structures were determined without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling under near native conditions.

scholarly journals S  (+)-Ketamine Suppresses Desensitization of γ-Aminobutyric Acid Type B Receptor-mediated Signaling by Inhibition of the Interaction of γ-Aminobutyric Acid Type B Receptors with G Protein–coupled Receptor Kinase 4 or 5

2011 ◽  
Vol 114 (2) ◽  
pp. 401-411 ◽  
Author(s):  
Yuko Ando ◽  
Minoru Hojo ◽  
Masato Kanaide ◽  
Masafumi Takada ◽  
Yuka Sudo ◽  
...  
Keyword(s):  
Complex Formation ◽  
G Protein ◽  
Protein Complex ◽  
Protein Complexes ◽  
Aminobutyric Acid ◽  
K Channel ◽  
Type B ◽  
Protein Complex Formation ◽  
Acid Type ◽  
G Protein Coupled

Background Intrathecal baclofen therapy is an established treatment for severe spasticity. However, long-term management occasionally results in the development of tolerance. One of the mechanisms of tolerance is desensitization of γ-aminobutyric acid type B receptor (GABABR) because of the complex formation of the GABAB2 subunit (GB2R) and G protein-coupled receptor kinase (GRK) 4 or 5. The current study focused on S(+)-ketamine, which reduces the development of morphine tolerance. This study was designed to investigate whether S(+)-ketamine affects the GABABR desensitization processes by baclofen. Methods The G protein-activated inwardly rectifying K channel currents induced by baclofen were recorded using Xenopus oocytes coexpressing G protein-activated inwardly rectifying K channel 1/2, GABAB1a receptor subunit, GB2R, and GRK. Translocation of GRKs 4 and 5 and protein complex formation of GB2R with GRKs were analyzed by confocal microscopy and fluorescence resonance energy transfer analysis in baby hamster kidney cells coexpressing GABAB1a receptor subunit, fluorescent protein-tagged GB2R, and GRKs. The formation of protein complexes of GB2R with GRKs was also determined by coimmunoprecipitation and Western blot analysis. Results Desensitization of GABABR-mediated signaling was suppressed by S(+)-ketamine in a concentration-dependent manner in the electrophysiologic assay. Confocal microscopy revealed that S(+)-ketamine inhibited translocation of GRKs 4 and 5 to the plasma membranes and protein complex formation of GB2R with the GRKs. Western blot analysis also showed that S(+)-ketamine inhibited the protein complex formation of GB2R with the GRKs. Conclusion S(+)-Ketamine suppressed the desensitization of GABABR-mediated signaling at least in part through inhibition of formation of protein complexes of GB2R with GRK 4 or 5.

Selective arrangement of vesicles on artificial lipid membrane by biotin-avidin interaction

2021 ◽  
Author(s):  
Kai Hashino ◽  
Daiya Mombayashi ◽  
Yuto Nakatani ◽  
Azusa Oshima ◽  
Masumi Yamaguchi ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Liquid Crystalline ◽  
Lipid Membrane ◽  
Unsaturated Lipid ◽  
Si Substrates ◽  
Phase Domain ◽  
Unsaturated Lipids ◽  
Lipid Mixtures ◽  
The Difference

Abstract Lipid bilayers suspended over microwells on Si substrates are promising platforms for nanobiodevices that mimic cell membranes. Using the biotin-avidin interaction, we have succeeded in selectively arranging vesicles on the freestanding region of a lipid bilayer. When ternary lipid mixtures of saturated lipid, unsaturated lipid, and cholesterol are used, they separate into liquid-order (Lo) and liquid-crystalline (Lα) domains. A freestanding lipid bilayer prefers the Lα-phase over the Lo-phase because of the difference in their flexibility. In addition, the type of biotinylated lipid determines whether it is localized in the Lα-phase domain or the Lo-phase domain. As a result, the biotinylated unsaturated lipids localized in the Lα-phase domain aggregate in the freestanding lipid bilayer, and vesicles labeled with biotin selectively bind to the freestanding lipid bilayer by the biotin-avidin interaction. This technique helps to introduce biomolecules into the freestanding lipid bilayer of nanobiodevices via vesicles.

scholarly journals Structure of a D2 dopamine receptor–G-protein complex in a lipid membrane

Nature ◽  
2020 ◽  
Vol 584 (7819) ◽  
pp. 125-129 ◽  
Author(s):  
Jie Yin ◽  
Kuang-Yui M. Chen ◽  
Mary J. Clark ◽  
Mahdi Hijazi ◽  
Punita Kumari ◽  
...  
Keyword(s):  
Dopamine Receptor ◽  
G Protein ◽  
Protein Complex ◽  
Lipid Membrane ◽  
D2 Dopamine Receptor

scholarly journals Dissecting the conformation of glycans and their interactions with proteins

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Sheng-Hung Wang ◽  
Tsai-Jung Wu ◽  
Chien-Wei Lee ◽  
John Yu
Keyword(s):  
Conformational Changes ◽  
Molecular Interactions ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Unmet Need ◽  
Protein Molecule ◽  
Structural Basis ◽  
Glycan Structure ◽  
Chronic Obstructive ◽  
Technology Platforms

Abstract The use of in silico strategies to develop the structural basis for a rational optimization of glycan-protein interactions remains a great challenge. This problem derives, in part, from the lack of technologies to quantitatively and qualitatively assess the complex assembling between a glycan and the targeted protein molecule. Since there is an unmet need for developing new sugar-targeted therapeutics, many investigators are searching for technology platforms to elucidate various types of molecular interactions within glycan-protein complexes and aid in the development of glycan-targeted therapies. Here we discuss three important technology platforms commonly used in the assessment of the complex assembly of glycosylated biomolecules, such as glycoproteins or glycosphingolipids: Biacore analysis, molecular docking, and molecular dynamics simulations. We will also discuss the structural investigation of glycosylated biomolecules, including conformational changes of glycans and their impact on molecular interactions within the glycan-protein complex. For glycoproteins, secreted protein acidic and rich in cysteine (SPARC), which is associated with various lung disorders, such as chronic obstructive pulmonary disease (COPD) and lung cancer, will be taken as an example showing that the core fucosylation of N-glycan in SPARC regulates protein-binding affinity with extracellular matrix collagen. For glycosphingolipids (GSLs), Globo H ceramide, an important tumor-associated GSL which is being actively investigated as a target for new cancer immunotherapies, will be used to demonstrate how glycan structure plays a significant role in enhancing angiogenesis in tumor microenvironments.

scholarly journals Structural Insights into the Process of GPCR-G Protein Complex Formation

Cell ◽  
2019 ◽  
Vol 177 (5) ◽  
pp. 1243-1251.e12 ◽  
Author(s):  
Xiangyu Liu ◽  
Xinyu Xu ◽  
Daniel Hilger ◽  
Philipp Aschauer ◽  
Johanna K.S. Tiemann ◽  
...  
Keyword(s):  
Complex Formation ◽  
G Protein ◽  
Protein Complex ◽  
Protein Complex Formation ◽  
Structural Insights

scholarly journals Structural basis of G protein–coupled receptor–G protein interactions

2010 ◽  
Vol 6 (7) ◽  
pp. 541-548 ◽  
Author(s):  
Jianxin Hu ◽  
Yan Wang ◽  
Xiaohong Zhang ◽  
John R Lloyd ◽  
Jian Hua Li ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Structural Basis ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Theoretical and computational modeling of peptide-lipid bilayer interaction studied by dynamic force spectroscopy

2019 ◽  
Author(s):  
◽  
Milica Utjesanovic
Keyword(s):  
Computational Modeling ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Lipid Membrane ◽  
Conformational Dynamics ◽  
Quantitative Description ◽  
Md Simulations ◽  
Dynamic Force ◽  
Detachment Rate

This thesis consists of three interrelated theoretical and computational modeling projects that investigate different aspects of peptide-lipid membrane interactions. (1) A general theoretical approach is formulated for the quantitative description of the detachment force distribution, P(F), and the corresponding force dependent detachment rate, k(F), of a peptide from a lipid bilayer, by assuming that peptide detachment from lipid membranes occurs stochastically along a few dominant diffusive pathways. Besides providing a consistent interpretation of the experimental data, the new method also predicts that k(F) exhibits catch-bond behavior (when, counter intuitively, the detachment rate decreases with increasing force). (2) The proposed multiple detachment pathways method is tested and validated for a particular peptide (SecA2-11) interacting with both zwitterionic POPC lipid and polar E. Coli membranes. Furthermore, molecular dynamics (MD) simulations are used to explored the conformational dynamics of SecA2-11 during its interaction with both POPC and anionic POPG lipid bilayers. (3) Finally, MD simulations are used to explore the conformational dynamics and energetics of the peptide melittin (MWT) and its diastereomer (MD4) interacting with POPC and POPG lipid bilayers. The obtained results provide further insight into the role of secondary structure in peptide-lipid bilayer interactions.

scholarly journals G-Protein-Coupled Receptor-Membrane Interactions Depend on the Receptor Activation state

2019 ◽  
Author(s):  
Apurba Bhattarai ◽  
Jinan Wang ◽  
Yinglong Miao
Keyword(s):  
G Protein ◽  
Lipid Bilayer ◽  
Protein Interactions ◽  
Membrane Lipids ◽  
Ischemia Reperfusion ◽  
Receptor Activation ◽  
Renal Diseases ◽  
Conformational States ◽  
Highly Correlated ◽  
G Protein Coupled

AbstractG-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and serve as primary targets of ∼1/3 of currently marketed drugs. In particular, adenosine A1 receptor (A1AR) is an important therapeutic target for treating cardiac ischemia-reperfusion injuries, neuropathic pain and renal diseases. As a prototypical GPCR, the A1AR is located within a phospholipid membrane bilayer and transmits cellular signals by changing between different conformational states. It is important to elucidate the lipid-protein interactions in order to understand the functional mechanism of GPCRs. Here, all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method were performed on both the inactive (antagonist bound) and active (agonist and G protein bound) A1AR, which was embedded in a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer. In the GaMD simulations, the membrane lipids played a key role in stabilizing different conformational states of the A1AR. Our simulations further identified important regions of the receptor that interacted distinctly with the lipids in highly correlated manner. Activation of the A1AR led to differential dynamics in the upper and lower leaflets of the lipid bilayer. In summary, GaMD enhanced simulations have revealed strongly coupled dynamics of the GPCR and lipids that depend on the receptor activation state.

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