29 results on '"Ouliana Panova"'
Search Results
2. The tethered peptide activation mechanism of adhesion GPCRs
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Ximena Barros-Álvarez, Robert M. Nwokonko, Alexander Vizurraga, Donna Matzov, Feng He, Makaía M. Papasergi-Scott, Michael J. Robertson, Ouliana Panova, Eliane Hadas Yardeni, Alpay B. Seven, Frank E. Kwarcinski, Hongyu Su, Maria Claudia Peroto, Justin G. Meyerowitz, Moran Shalev-Benami, Gregory G. Tall, and Georgios Skiniotis
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Multidisciplinary - Published
- 2022
3. Time-resolved cryo-EM of G protein activation by a GPCR
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Makaía M. Papasergi-Scott, Guillermo Pérez-Hernández, Hossein Batebi, Yang Gao, Gözde Eskici, Alpay B. Seven, Ouliana Panova, Daniel Hilger, Marina Casiraghi, Feng He, Luis Maul, Peter Gmeiner, Brian K. Kobilka, Peter W. Hildebrand, and Georgios Skiniotis
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Article - Abstract
SummaryG protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating the exchange of guanine nucleotide in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G protein complex. Using variability analysis to monitor the transitions of the stimulatory Gs protein in complex with the β2-adrenergic receptor (β2AR) at short sequential time points after GTP addition, we identified the conformational trajectory underlying G protein activation and functional dissociation from the receptor. Twenty transition structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of events driving G protein activation upon GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα Switch regions and the α5 helix that weaken the G protein-receptor interface. Molecular dynamics (MD) simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP upon closure of the alpha-helical domain (AHD) against the nucleotide-bound Ras-homology domain (RHD) correlates with irreversible α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signaling events.
- Published
- 2023
4. The oxytocin signaling complex reveals a molecular switch for cation dependence
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Justin G. Meyerowitz, Michael J. Robertson, Ximena Barros-Álvarez, Ouliana Panova, Robert M. Nwokonko, Yang Gao, and Georgios Skiniotis
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Structural Biology ,Molecular Biology - Published
- 2022
5. Plasticity in ligand recognition at somatostatin receptors
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Georgios Skiniotis, Justin G. Meyerowitz, Kenneth W. Borrelli, Ouliana Panova, and Michael J. Robertson
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chemistry.chemical_classification ,Drug discovery ,Somatostatin receptor ,Octreotide ,Peptide ,Ligand (biochemistry) ,Ligands ,Cell biology ,Somatostatin ,chemistry ,Structural Biology ,Extracellular ,medicine ,Somatostatin receptor 2 ,Receptors, Somatostatin ,Molecular Biology ,medicine.drug - Abstract
Somatostatin is a signaling peptide that plays a pivotal and wide-ranging role in physiologic processes relating to metabolism and growth through its actions at somatostatin receptors (SSTRs). Members of the somatostatin receptor subfamily, particularly SSTR2, are key drug targets for neuroendocrine neoplasms, with several synthetic peptide agonists currently in clinical use. Here, we show the cryogenic electron microscopy (cryo-EM) structures of active-state SSTR2 in complex with heterotrimeric Gi protein and either the endogenous ligand SST14 or the FDA-approved drug octreotide.Complemented by biochemical assays and molecular dynamics simulations, these structures reveal key details of ligand recognition, receptor activation, and subtype-selectivity at somatostatin receptors. We find that SSTR ligand recognition is highly diverse, as demonstrated by ligand-induced conformational changes in ECL2, substantial sequence divergence across subtypes in extracellular regions, and loss of ligand binding upon several structurally homologous substitutions between subtypes. Despite this complexity, we rationalize several previously described sources of SSTR subtype selectivity and identify an additional key interaction for SSTR2/3/5 specific binding. These results shed light on the basis of ligand recognition by somatostatin receptors and provide valuable insights for structure-based drug discovery at these targets.
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- 2022
6. Structure determination of inactive-state GPCRs with a universal nanobody
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Michael J, Robertson, Makaía M, Papasergi-Scott, Feng, He, Alpay B, Seven, Justin G, Meyerowitz, Ouliana, Panova, Maria Claudia, Peroto, Tao, Che, and Georgios, Skiniotis
- Abstract
Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.
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- 2022
7. Enhancing the chameleon grid range to unlock faster and more precise sample optimisation
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Miriam Weckener, Ouliana Panova, Herve Vandekerckhove, Nicolas Marro, Russell King, Paul Thaw, Daniel K. Clare, and Michele C. Darrow
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Biophysics - Published
- 2023
8. Asymmetric activation of the calcium-sensing receptor homodimer
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Sabrina N. Rahman, Michael J. Robertson, Alpay B. Seven, Fadil M. Hannan, Jesper Mosolff Mathiesen, Ouliana Panova, Rajesh V. Thakker, Yang Gao, Georgios Skiniotis, Chensong Zhang, Hans Bräuner-Osborne, and Justin G. Meyerowitz
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Models, Molecular ,Calcimimetic ,Protomer ,Article ,Substrate Specificity ,03 medical and health sciences ,0302 clinical medicine ,Heterotrimeric G protein ,Humans ,Receptor ,030304 developmental biology ,G protein-coupled receptor ,0303 health sciences ,Multidisciplinary ,Chemistry ,Cryoelectron Microscopy ,Rational design ,Calcilytic ,Biophysics ,Calcium ,Calcium-sensing receptor ,Protein Multimerization ,Peptides ,Receptors, Calcium-Sensing ,030217 neurology & neurosurgery ,Protein Binding - Abstract
The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders1. CaSR is a family C G-protein-coupled receptor2 that functions as an obligate homodimer, with each protomer composed of a Ca2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor. Cryo-EM structures of human calcium-sensing receptor reveal intrinsic asymmetry in the receptor homodimer upon activation that is stabilized by calcimimetic drugs adopting distinct poses in the two protomers, priming one protomer for G-protein coupling.
- Published
- 2022
9. Atypical structural snapshots of human cytomegalovirus GPCR interactions with host G proteins
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Naotaka Tsutsumi, Shoji Maeda, Qianhui Qu, Martin Vögele, Kevin M. Jude, Carl-Mikael Suomivuori, Ouliana Panova, Deepa Waghray, Hideaki E. Kato, Andrew Velasco, Ron O. Dror, Georgios Skiniotis, Brian K. Kobilka, and K. Christopher Garcia
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Mammals ,Multidisciplinary ,Cryoelectron Microscopy ,SciAdv r-articles ,Cytomegalovirus ,Viral Proteins ,Structural Biology ,GTP-Binding Proteins ,Virology ,Animals ,Humans ,Receptors, Chemokine ,Biomedicine and Life Sciences ,Health and Medicine ,Research Article - Abstract
Human cytomegalovirus (HCMV) encodes G protein–coupled receptors (GPCRs) US28 and US27, which facilitate viral pathogenesis through engagement of host G proteins. Here we report cryo–electron microscopy structures of US28 and US27 forming nonproductive and productive complexes with Gi and Gq, respectively, exhibiting unusual features with functional implications. The “orphan” GPCR US27 lacks a ligand-binding pocket and has captured a guanosine diphosphate–bound inactive Gi through a tenuous interaction. The docking modes of CX3CL1-US28 and US27 to Gi favor localization to endosome-like curved membranes, where US28 and US27 can function as nonproductive Gi sinks to attenuate host chemokine-dependent Gi signaling. The CX3CL1-US28-Gq/11 complex likely represents a trapped intermediate during productive signaling, providing a view of a transition state in GPCR–G protein coupling for signaling. Our collective results shed new insight into unique G protein–mediated HCMV GPCR structural mechanisms, compared to mammalian GPCR counterparts, for subversion of host immunity., Description, The HCMV GPCR-human G protein structures reveal unusual host-pathogen interactions likely critical for HCMV’s pathogenesis.
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- 2022
10. Signaling Snapshots of 5-HT 2BR Activated by the Prototypical Psychedelic LSD
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Can Cao, Ximena Barros-Álvarez, Shicheng Zhang, Kuglae Kim, Marc A. Dämgen, Ouliana Panova, Carl-Mikael Suomivuori, Jonathan Fay, Xiaofang Zhong, Brian E. Krumm, Ryan H. Gumpper, Alpay B. Seven, Michael J. Robertson, Nevan J. Krogan, Ruth Hüttenhain, David E. Nichols, Ron O. Dror, Georgios Skiniotis, and Bryan Roth
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History ,Polymers and Plastics ,Business and International Management ,Industrial and Manufacturing Engineering - Published
- 2022
11. Structures of Ric-8B in Complex With Gα Protein Folding Clients Reveal Isoform Specificity Mechanisms
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Makaía M. Papasergi-Scott, Frank E. Kwarcinski, Maiya Yu, Ouliana Panova, Ann M. Ovrutsky, Georgios Skiniotis, and Gregory G. Tall
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History ,Polymers and Plastics ,Structural Biology ,Business and International Management ,Molecular Biology ,Industrial and Manufacturing Engineering - Published
- 2022
12. Structure Determination of Inactive-State GPCRs with a Universal Nanobody
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Michael J. Robertson, Makaía Papasergi-Scott, Feng He, Alpay B. Seven, Justin G. Meyerowitz, Ouliana Panova, Maria Claudia Peroto, Tao Che, and Georgios Skiniotis
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Membrane protein ,Structural Biology ,Chemistry ,Drug discovery ,Biophysics ,Somatostatin receptor 2 ,Receptor ,Molecular Biology ,G protein-coupled receptor - Abstract
SummaryCryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. However, despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of different inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained the structure of human neurotensin 1 receptor (NTSR1) bound to antagonist SR48692, of µ-opioid receptor (MOR) bound to the clinical antagonist alvimopan, as well as the structures of the previously uncharacterized somatostatin receptor 2 (SSTR2) in the apo state and histamine receptor 2 (H2R) bound to the H2 blocker famotidine. Each of these structures yields novel insights into ligand binding and specificity. We expect this rapid, straightforward approach to facilitate the broad structural exploration of GPCR inactive states without the need for extensive engineering and crystallization.
- Published
- 2021
13. The oxytocin signaling complex reveals a molecular switch for cation dependence
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Justin G, Meyerowitz, Michael J, Robertson, Ximena, Barros-Álvarez, Ouliana, Panova, Robert M, Nwokonko, Yang, Gao, and Georgios, Skiniotis
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Receptors, Vasopressin ,Pregnancy ,Receptors, Oxytocin ,Cations ,Animals ,Female ,Magnesium ,Oxytocin ,Signal Transduction - Abstract
Oxytocin (OT) and vasopressin (AVP) are conserved peptide signaling hormones that are critical for diverse processes including osmotic homeostasis, reproduction, lactation and social interaction. OT acts through the oxytocin receptor (OTR), a magnesium-dependent G protein-coupled receptor that is a therapeutic target for treatment of postpartum hemorrhage, dysfunctional labor and autism. However, the molecular mechanisms that underlie OTR activation by OT and the dependence on magnesium remain unknown. Here we present the wild-type active-state structure of human OTR bound to OT and miniG
- Published
- 2021
14. Signaling snapshots of a serotonin receptor activated by the prototypical psychedelic LSD
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Can Cao, Ximena Barros-Álvarez, Shicheng Zhang, Kuglae Kim, Marc A. Dämgen, Ouliana Panova, Carl-Mikael Suomivuori, Jonathan F. Fay, Xiaofang Zhong, Brian E. Krumm, Ryan H. Gumpper, Alpay B. Seven, Michael J. Robertson, Nevan J. Krogan, Ruth Hüttenhain, David E. Nichols, Ron O. Dror, Georgios Skiniotis, and Bryan L. Roth
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Lysergic Acid Diethylamide ,Serotonin ,Receptors, Serotonin ,General Neuroscience ,Hallucinogens ,beta-Arrestins - Abstract
Serotonin (5-hydroxytryptamine [5-HT]) 5-HT2-family receptors represent essential targets for lysergic acid diethylamide (LSD) and all other psychedelic drugs. Although the primary psychedelic drug effects are mediated by the 5-HT
- Published
- 2022
15. Bespoke library docking for 5-HT
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Anat Levit, Kaplan, Danielle N, Confair, Kuglae, Kim, Ximena, Barros-Álvarez, Ramona M, Rodriguiz, Ying, Yang, Oh Sang, Kweon, Tao, Che, John D, McCorvy, David N, Kamber, James P, Phelan, Luan Carvalho, Martins, Vladimir M, Pogorelov, Jeffrey F, DiBerto, Samuel T, Slocum, Xi-Ping, Huang, Jain Manish, Kumar, Michael J, Robertson, Ouliana, Panova, Alpay B, Seven, Autumn Q, Wetsel, William C, Wetsel, John J, Irwin, Georgios, Skiniotis, Brian K, Shoichet, Bryan L, Roth, and Jonathan A, Ellman
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Small Molecule Libraries ,Mice ,Pyrrolidines ,Fluoxetine ,Cryoelectron Microscopy ,Hallucinogens ,Animals ,Receptor, Serotonin, 5-HT2A ,Ligands ,Antidepressive Agents - Abstract
There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally
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- 2021
16. Optimization of CryoEM Sample Preparation: A New Freezing Strategy to Reduce the Time to Structure Loop
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Ouliana Panova, Herve Vandekerckhove, Nicolas Marro, Stefanie Rajasooriya, Tim Booth, and Michele C Darrow
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Instrumentation - Published
- 2022
17. Author Correction: Plasticity in ligand recognition at somatostatin receptors
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Michael J. Robertson, Justin G. Meyerowitz, Ouliana Panova, Kenneth Borrelli, and Georgios Skiniotis
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Structural Biology ,Molecular Biology - Published
- 2022
18. Diffraction imaging of nanocrystalline structures in organic semiconductor molecular thin films
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Luke Balhorn, Karen C. Bustillo, Andrew M. Minor, Nitash P. Balsara, Ouliana Panova, Alberto Salleo, Colin Ophus, and Christopher J. Takacs
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Diffraction ,Materials science ,Mechanical Engineering ,02 engineering and technology ,General Chemistry ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,01 natural sciences ,Nanocrystalline material ,0104 chemical sciences ,law.invention ,Organic semiconductor ,Reciprocal lattice ,Mechanics of Materials ,Chemical physics ,law ,Transmission electron microscopy ,General Materials Science ,Grain boundary ,Thin film ,Electron microscope ,0210 nano-technology - Abstract
The properties of organic solids depend on their structure and morphology, yet direct imaging using conventional electron microscopy methods is hampered by the complex internal structure of these materials and their sensitivity to electron beams. Here, we manage to observe the nanocrystalline structure of two organic molecular thin-film systems using transmission electron microscopy by employing a scanning nanodiffraction method that allows for full access to reciprocal space over the size of a spatially localized probe (~2 nm). The morphologies revealed by this technique vary from grains with pronounced segmentation of the structure-characterized by sharp grain boundaries and overlapping domains-to liquid-crystal structures with crystalline orientations varying smoothly over all possible rotations that contain disclinations representing singularities in the director field. The results show how structure-property relationships can be visualized in organic systems using techniques previously only available for hard materials such as metals and ceramics.
- Published
- 2019
19. Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor
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Michael J. Robertson, Alpay B. Seven, Ouliana Panova, Tao Che, Jeffrey F. DiBerto, Kuglae Kim, Brian K. Shoichet, Brian E. Krumm, David E. Nichols, Georgios Skiniotis, Jiankun Lyu, Daniel Wacker, and Bryan L. Roth
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Protein Conformation, alpha-Helical ,Protein Conformation ,Methiothepin ,Gene Expression ,Crystallography, X-Ray ,Ligands ,Medical and Health Sciences ,Psilocybin ,LSD ,Substance Misuse ,0302 clinical medicine ,GPCR ,Models ,5-HT2A ,structural biology ,2.1 Biological and endogenous factors ,Receptor, Serotonin, 5-HT2A ,Aetiology ,Lysergic acid diethylamide ,0303 health sciences ,Crystallography ,Depression ,Biological Sciences ,Ligand (biochemistry) ,GTP-Binding Protein alpha Subunits ,Recombinant Proteins ,Mental Health ,signal transduction ,medicine.drug ,Receptor ,Hallucinogen ,Serotonin ,Chemical ,Biology ,Spodoptera ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,medicine ,Inverse agonist ,Animals ,Humans ,5-HT receptor ,030304 developmental biology ,G protein-coupled receptor ,Gq-G11 ,sertotonin receptor ,Cryoelectron Microscopy ,alpha-Helical ,Brain Disorders ,Lysergic Acid Diethylamide ,HEK293 Cells ,Models, Chemical ,Structural biology ,psychedelic ,Mutation ,Hallucinogens ,X-Ray ,GTP-Binding Protein alpha Subunits, Gq-G11 ,Neuroscience ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions—both therapeutic and hallucinogenic—are not understood, although activation of the 5-HT(2A) serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH—a prototypical hallucinogen—in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.
- Published
- 2020
20. Structures of metabotropic GABAB receptor
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Makaía M. Papasergi-Scott, Alpay B. Seven, Ouliana Panova, Michael J. Robertson, Jesper Mosolff Mathiesen, and Georgios Skiniotis
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0301 basic medicine ,Models, Molecular ,G protein ,Neurotransmission ,GABAB receptor ,Ligands ,Article ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Heterotrimeric G protein ,Humans ,Receptor ,Phospholipids ,Calcium signaling ,030304 developmental biology ,G protein-coupled receptor ,0303 health sciences ,Multidisciplinary ,Binding Sites ,Chemistry ,Cell Membrane ,Cryoelectron Microscopy ,Cell biology ,Protein Subunits ,Transmembrane domain ,030104 developmental biology ,Metabotropic receptor ,nervous system ,Receptors, GABA-B ,Receptors, Glutamate ,Protein Multimerization ,Signal transduction ,GABA-B Receptor Antagonists ,Hydrophobic and Hydrophilic Interactions ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Stimulation of the metabotropic GABAB receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology1. GABAB belongs to family C of the G-protein-coupled receptors, which operate as dimers to transform synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins2,3. However, GABAB is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits4,5. Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABAB has an essential role in relaying structural transitions by ordering the linker that connects the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of each of the subunits of GABAB caps and interacts with the hydrophilic head of a phospholipid that occupies the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G proteins. These results provide a starting framework through which to decipher the mechanistic modes of signal transduction mediated by GABAB dimers, and have important implications for rational drug design that targets these receptors. Cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors, combined with cellular signalling assays, shed light on the mechanisms that underpin signal transduction mediated by these receptors.
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- 2020
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21. Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6
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Gozde Eskici, Richard A. Cerione, Georgios Skiniotis, Ouliana Panova, Yang Gao, Sekar Ramachandran, Frédéric Poitevin, and Alpay B. Seven
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Models, Molecular ,G protein ,Biology ,Inhibitory postsynaptic potential ,Photoreceptor cell ,Article ,Enzyme catalysis ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Vardenafil Dihydrochloride ,Catalytic Domain ,Heterotrimeric G protein ,medicine ,Animals ,Transducin ,Receptor ,Molecular Biology ,030304 developmental biology ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,0303 health sciences ,Α subunit ,Effector ,Chemistry ,030302 biochemistry & molecular biology ,Phosphodiesterase ,Cell Biology ,3. Good health ,medicine.anatomical_structure ,Biocatalysis ,Biophysics ,Cattle ,Guanosine Triphosphate ,sense organs ,030217 neurology & neurosurgery ,Protein Binding ,Signal Transduction ,Visual phototransduction - Abstract
SUMMARYHeterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is comprised of the GTP-bound transducin α subunit (GαT·GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis to transmit signals to the optic nerve. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GαT. The structure reveals two GαT·GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDEγ subunits, driving extensive rearrangements to relieve all inhibitory constraints on enzyme catalysis. Analysis of the conformational ensemble in the cryoEM data highlights the dynamic nature of the contacts between the two GαT·GTP subunits and PDE6 that support an alternating-site catalytic mechanism.
- Published
- 2020
22. Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells
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Georgios Skiniotis, Yamuna Kalyani Mathiharan, Patrick J. Lupardus, Leon Su, Christoph Thomas, Jamie B. Spangler, Kevin Jude, Lauren Kate Ely, Caleb R. Glassman, K. Christopher Garcia, and Ouliana Panova
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Models, Molecular ,medicine.medical_treatment ,Protein subunit ,T-Lymphocytes ,Biology ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Epitopes ,Structure-Activity Relationship ,0302 clinical medicine ,Neoplasms ,medicine ,Interleukin 23 ,Animals ,Humans ,Interferon gamma ,Amino Acid Sequence ,Receptor ,Protein Structure, Quaternary ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Interleukin-12 Subunit p40 ,Cryoelectron Microscopy ,Immunity ,Receptors, Interleukin-12 ,Receptors, Interleukin ,Interleukin-12 ,Cell biology ,Killer Cells, Natural ,Mice, Inbred C57BL ,Cytokine ,HEK293 Cells ,Structural biology ,Interleukin 12 ,Female ,030217 neurology & neurosurgery ,CD8 ,medicine.drug ,Signal Transduction - Abstract
Summary Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rβ1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rβ1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rβ2/IL-12Rβ1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rβ1 directly engages the common p40 subunit. We targeted the shared IL-12Rβ1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.
- Published
- 2020
23. Solution-grown crystals of precise acid- and ion-containing polyethylenes
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Ouliana Panova, Karen I. Winey, Lu Yan, Karen C. Bustillo, and Andrew M. Minor
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chemistry.chemical_classification ,Materials science ,Polymers and Plastics ,Geminal ,Carboxylic acid ,Organic Chemistry ,Ionic bonding ,02 engineering and technology ,Polymer ,Polyethylene ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,0104 chemical sciences ,Crystallography ,chemistry.chemical_compound ,symbols.namesake ,chemistry ,Phase (matter) ,Materials Chemistry ,symbols ,Orthorhombic crystal system ,0210 nano-technology ,Raman spectroscopy - Abstract
Crystals of precise acid- and ion-containing polyethylenes were prepared from solution. While large pendant groups on polyethylene backbones are typically excluded from the crystalline domain, the precisely-placed acid and ionic functional groups are accommodated into the solution-grown crystals. Polyethylene containing carboxylic acid pendant groups on every 21st carbon atom (p21AA) grows into rectangular shaped crystals with an average thickness of 9 nm, which is 3–4 times the all-trans chain length between the functional groups. This thickness indicates that the carboxylic acid groups are incorporated within the crystals. Electron diffraction images and Raman spectra indicate that p21AA backbones are hexagonally packed with more gauche chain conformations than the polyethylene orthorhombic phase. The precise polyethylene containing geminal carboxylic acid groups placed every 21 carbon atoms (p21gAA) forms irregular-shaped crystals with an average thickness 8 nm. Similar to p21AA, p21gAA incorporates the geminal acid groups into its solution-grown crystals. Surprisingly, the polymer with imidazolium bromide groups on every 21st carbon (p21ImBr) produces remarkably large crystals with thicknesses of 200–900 nm, and widths of 6–12 μm. Finally, we propose a multi-layer stack of adjacent reentry structures that is consistent with the incorporation of large functional groups into the solution-growth crystals of these precise polyethylenes. Interestingly, this stacked structure is distinct from our recent results in melt-crystallized p21AA, where the layers are nearly transverse to the lamellae.
- Published
- 2018
24. Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction
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Jose A. Rodriguez, Calina Glynn, Andrew M. Minor, David R. Boyer, Ouliana Panova, Karen C. Bustillo, Kevin Canton Mancia, Marcus Gallagher-Jones, Chih-Te Zee, Jim Ciston, and Colin Ophus
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Diffraction ,Materials science ,Scanning electron microscope ,Medicine (miscellaneous) ,Physics::Optics ,Bioengineering ,02 engineering and technology ,Crystal structure ,Article ,General Biochemistry, Genetics and Molecular Biology ,Mosaicity ,Crystal ,03 medical and health sciences ,Crystallinity ,Theoretical ,Models ,Nanotechnology ,lcsh:QH301-705.5 ,030304 developmental biology ,0303 health sciences ,Models, Theoretical ,021001 nanoscience & nanotechnology ,lcsh:Biology (General) ,Electron diffraction ,Chemical physics ,Nanoparticles ,0210 nano-technology ,General Agricultural and Biological Sciences ,Protein crystallization ,Peptides - Abstract
Changes in lattice structure across sub-regions of protein crystals are challenging to assess when relying on whole crystal measurements. Because of this difficulty, macromolecular structure determination from protein micro and nanocrystals requires assumptions of bulk crystallinity and domain block substructure. Here we map lattice structure across micron size areas of cryogenically preserved three−dimensional peptide crystals using a nano-focused electron beam. This approach produces diffraction from as few as 1500 molecules in a crystal, is sensitive to crystal thickness and three−dimensional lattice orientation. Real-space maps reconstructed from unsupervised classification of diffraction patterns across a crystal reveal regions of crystal order/disorder and three−dimensional lattice tilts on the sub-100nm scale. The nanoscale lattice reorientation observed in the micron-sized peptide crystal lattices studied here provides a direct view of their plasticity. Knowledge of these features facilitates an improved understanding of peptide assemblies that could aid in the determination of structures from nano- and microcrystals by single or serial crystal electron diffraction., Marcus Gallagher-Jones et al. use 4DSTEM to reconstruct real-space maps of single peptide nanocrystals at cryogenic temperature. They identify regions of crystal order/disorder providing new insights into crystal mosaicity.
- Published
- 2019
25. Diffraction imaging of nanocrystalline structures in organic semiconductor molecular thin films
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Ouliana, Panova, Colin, Ophus, Christopher J, Takacs, Karen C, Bustillo, Luke, Balhorn, Alberto, Salleo, Nitash, Balsara, and Andrew M, Minor
- Abstract
The properties of organic solids depend on their structure and morphology, yet direct imaging using conventional electron microscopy methods is hampered by the complex internal structure of these materials and their sensitivity to electron beams. Here, we manage to observe the nanocrystalline structure of two organic molecular thin-film systems using transmission electron microscopy by employing a scanning nanodiffraction method that allows for full access to reciprocal space over the size of a spatially localized probe (~2 nm). The morphologies revealed by this technique vary from grains with pronounced segmentation of the structure-characterized by sharp grain boundaries and overlapping domains-to liquid-crystal structures with crystalline orientations varying smoothly over all possible rotations that contain disclinations representing singularities in the director field. The results show how structure-property relationships can be visualized in organic systems using techniques previously only available for hard materials such as metals and ceramics.
- Published
- 2018
26. Orientation mapping of semicrystalline polymers using scanning electron nanobeam diffraction
- Author
-
Andrew M. Minor, Ouliana Panova, Colin Ophus, Nitash P. Balsara, Mahesh P. Bhatt, Karen C. Bustillo, and X. Chelsea Chen
- Subjects
Diffraction ,Materials science ,Reflection high-energy electron diffraction ,Orientation (computer vision) ,business.industry ,Scanning electron microscope ,General Physics and Astronomy ,02 engineering and technology ,Cell Biology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,0104 chemical sciences ,Crystallinity ,Optics ,Structural Biology ,General Materials Science ,Polymer blend ,Crystallite ,0210 nano-technology ,business ,Electron backscatter diffraction - Abstract
We demonstrate a scanning electron nanobeam diffraction technique that can be used for mapping the size and distribution of nanoscale crystalline regions in a polymer blend. In addition, it can map the relative orientation of crystallites and the degree of crystallinity of the material. The model polymer blend is a 50:50w/w mixture of semicrystalline poly(3-hexylthiophene-2,5-diyl) (P3HT) and amorphous polystyrene (PS). The technique uses a scanning electron beam to raster across the sample and acquires a diffraction image at each probe position. Through image alignment and filtering, the diffraction image dataset enables mapping of the crystalline regions within the scanned area and construction of an orientation map.
- Published
- 2016
27. Nanobeam Scanning Diffraction for Orientation Mapping of Polymers
- Author
-
Andrew M. Minor, Ouliana Panova, Colin Ophus, Christopher J. Takacs, Nitash P. Balsara, X. Chelsea Chen, Jim Ciston, and Karen C. Bustillo
- Subjects
010302 applied physics ,chemistry.chemical_classification ,Diffraction ,Materials science ,business.industry ,02 engineering and technology ,Polymer ,Orientation (graph theory) ,021001 nanoscience & nanotechnology ,01 natural sciences ,Optics ,chemistry ,0103 physical sciences ,0210 nano-technology ,business ,Instrumentation - Published
- 2017
28. Development of Diffraction Scanning Techniques for Beam Sensitive Polymers
- Author
-
Andrew M. Minor, Edward B. Trigg, Karen I. Winey, Lu Yan, Karen C. Bustillo, X. Chelsea Chen, Nitash P. Balsara, Ouliana Panova, and Christoph Gammer
- Subjects
Diffraction ,chemistry.chemical_classification ,Materials science ,business.industry ,02 engineering and technology ,Polymer ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,0104 chemical sciences ,chemistry ,Optoelectronics ,Development (differential geometry) ,0210 nano-technology ,business ,Instrumentation ,Beam (structure) - Published
- 2016
29. Nanobeam Scanning Diffraction for Orientation Mapping of Polymers.
- Author
-
Bustillo, Karen C., Ouliana Panova, Chen, X. Chelsea, Takacs, Christopher J., Ciston, Jim, Ophus, Colin, Balsara, Nitash P., and Minor, Andrew M.
- Published
- 2017
- Full Text
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