104 results on '"Deupi X"'
Search Results
2. Charge-charge and cation-π interactions in ligand binding to G protein-coupled receptors
- Author
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Dölker, N., Deupi, X., Pardo, L., and Campillo, M.
- Published
- 2007
- Full Text
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3. Selective hydrolysis of 2,4-diaminopyrimidine systems: a theoretical and experimental insight into an old rule
- Author
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Teixido, J., Borrell, J.I., Colominas, C., Deupi, X., Matallana, J.L., Falco, J.L., and Martinez-Teipel, B.
- Subjects
Pyrimidines -- Research ,Hydrolysis -- Methods ,Chemical reactions -- Evaluation ,Chemistry, Organic -- Research ,Biological sciences ,Chemistry - Abstract
The protocol for the synthesis of oxo-substituted pyrimidines by the hydrolysis of the amino groups in 2,4-diaminopyrimidine systems is reexamined. Analysis suggests that nitrogen linked to C6 of the 2,4-diaminopyrimidine ring alters the hydrolysis site, yielding 4-amino-2-oxypyrimidine isomer.
- Published
- 2001
4. Femtosecond to millisecond structural changes in a light-driven sodium pump: 1ns+16ns structure of KR2 with extrapolated, light and dark datasets
- Author
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Skopintsev, P., primary, Ehrenberg, D., additional, Weinert, T., additional, James, D., additional, Kar, R., additional, Johnson, P., additional, Ozerov, D., additional, Furrer, A., additional, Martiel, I., additional, Dworkowski, F., additional, Nass, K., additional, Knopp, G., additional, Cirelli, C., additional, Gashi, D., additional, Mous, S., additional, Wranik, M., additional, Gruhl, T., additional, Kekilli, D., additional, Bruenle, S., additional, Deupi, X., additional, Schertler, G.F.X., additional, Benoit, R., additional, Panneels, V., additional, Nogly, P., additional, Schapiro, I., additional, Milne, C., additional, Heberle, J., additional, and Standfuss, J., additional
- Published
- 2020
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5. Femtosecond to millisecond structural changes in a light-driven sodium pump: Dark structure in acidic conditions
- Author
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Skopintsev, P., primary, Ehrenberg, D., additional, Weinert, T., additional, James, D., additional, Kar, R., additional, Johnson, P., additional, Ozerov, D., additional, Furrer, A., additional, Martiel, I., additional, Dworkowski, F., additional, Nass, K., additional, Knopp, G., additional, Cirelli, C., additional, Gashi, D., additional, Mous, S., additional, Wranik, M., additional, Gruhl, T., additional, Kekilli, D., additional, Bruenle, S., additional, Deupi, X., additional, Schertler, G.F.X., additional, Benoit, R., additional, Panneels, V., additional, Nogly, P., additional, Schapiro, I., additional, Milne, C., additional, Heberle, J., additional, and Standfuss, J., additional
- Published
- 2020
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6. Femtosecond to millisecond structural changes in a light-driven sodium pump: 30us+150us structure of KR2 with extrapolated, light and dark datasets
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Skopintsev, P., primary, Ehrenberg, D., additional, Weinert, T., additional, James, D., additional, Kar, R., additional, Johnson, P., additional, Ozerov, D., additional, Furrer, A., additional, Martiel, I., additional, Dworkowski, F., additional, Nass, K., additional, Knopp, G., additional, Cirelli, C., additional, Gashi, D., additional, Mous, S., additional, Wranik, M., additional, Gruhl, T., additional, Kekilli, D., additional, Bruenle, S., additional, Deupi, X., additional, Schertler, G.F.X., additional, Benoit, R., additional, Panneels, V., additional, Nogly, P., additional, Schapiro, I., additional, Milne, C., additional, Heberle, J., additional, and Standfuss, J., additional
- Published
- 2020
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7. Femtosecond to millisecond structural changes in a light-driven sodium pump: 800fs+2ps structure of KR2 with extrapolated, light and dark datasets
- Author
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Skopintsev, P., primary, Ehrenberg, D., additional, Weinert, T., additional, James, D., additional, Kar, R., additional, Johnson, P., additional, Ozerov, D., additional, Furrer, A., additional, Martiel, I., additional, Dworkowski, F., additional, Nass, K., additional, Knopp, G., additional, Cirelli, C., additional, Gashi, D., additional, Mous, S., additional, Wranik, M., additional, Gruhl, T., additional, Kekilli, D., additional, Bruenle, S., additional, Deupi, X., additional, Schertler, G.F.X., additional, Benoit, R., additional, Panneels, V., additional, Nogly, P., additional, Schapiro, I., additional, Milne, C., additional, Heberle, J., additional, and Standfuss, J., additional
- Published
- 2020
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8. Femtosecond to millisecond structural changes in a light-driven sodium pump: 20ms structure of KR2 with extrapolated, light and dark datasets
- Author
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Skopintsev, P., primary, Ehrenberg, D., additional, Weinert, T., additional, James, D., additional, Kar, R., additional, Johnson, P., additional, Ozerov, D., additional, Furrer, A., additional, Martiel, I., additional, Dworkowski, F., additional, Nass, K., additional, Knopp, G., additional, Cirelli, C., additional, Gashi, D., additional, Mous, S., additional, Wranik, M., additional, Gruhl, T., additional, Kekilli, D., additional, Bruenle, S., additional, Deupi, X., additional, Schertler, G.F.X., additional, Benoit, R., additional, Panneels, V., additional, Nogly, P., additional, Schapiro, I., additional, Milne, C., additional, Heberle, J., additional, and Standfuss, J., additional
- Published
- 2020
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9. Femtosecond to millisecond structural changes in a light-driven sodium pump: Dark structure in neutral conditions with attached light datasets at 800fs, 2ps, 100ps, 1ns, 16ns, 1us, 30us, 150us, 1ms and 20ms
- Author
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Skopintsev, P., primary, Ehrenberg, D., additional, Weinert, T., additional, James, D., additional, Kar, R., additional, Johnson, P., additional, Ozerov, D., additional, Furrer, A., additional, Martiel, I., additional, Dworkowski, F., additional, Nass, K., additional, Knopp, G., additional, Cirelli, C., additional, Gashi, D., additional, Mous, S., additional, Wranik, M., additional, Gruhl, T., additional, Kekilli, D., additional, Bruenle, S., additional, Deupi, X., additional, Schertler, G.F.X., additional, Benoit, R., additional, Panneels, V., additional, Nogly, P., additional, Schapiro, I., additional, Milne, C., additional, Heberle, J., additional, and Standfuss, J., additional
- Published
- 2020
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10. Rhodopsin-Gi protein complex
- Author
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Tsai, C.-J., primary, Marino, J., additional, Adaixo, R.J., additional, Pamula, F., additional, Muehle, J., additional, Maeda, S., additional, Flock, T., additional, Taylor, N.M.I., additional, Mohammed, I., additional, Matile, H., additional, Dawson, R.J.P., additional, Deupi, X., additional, Stahlberg, H., additional, and Schertler, G.F.X., additional
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- 2019
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11. Antibody FAB fragment targeting Gi protein heterotrimer
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Tsai, C.-J., primary, Muehle, J., additional, Pamula, F., additional, Dawson, R.J.P., additional, Maeda, S., additional, Deupi, X., additional, and Schertler, G.F.X., additional
- Published
- 2019
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12. Crystal structure of Jumping Spider Rhodopsin-1 bound to 9-cis retinal
- Author
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Varma, N., primary, Mutt, E., additional, Muehle, J., additional, Panneels, V., additional, Terakita, A., additional, Deupi, X., additional, Nogly, P., additional, Schertler, F.X.G., additional, and Lesca, E., additional
- Published
- 2019
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13. Crystal structure of the rhodopsin-mini-Go complex
- Author
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Tsai, C.-J., primary, Weinert, T., additional, Muehle, J., additional, Pamula, F., additional, Nehme, R., additional, Flock, T., additional, Nogly, P., additional, Edwards, P.C., additional, Carpenter, B., additional, Gruhl, T., additional, Ma, P., additional, Deupi, X., additional, Standfuss, J., additional, Tate, C.G., additional, and Schertler, G.F.X., additional
- Published
- 2018
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14. Crystal Structure of T94I rhodopsin mutant
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Singhal, A., primary, Guo, Y., additional, Matkovic, M., additional, Schertler, G., additional, Deupi, X., additional, Yan, E., additional, and Standfuss, J., additional
- Published
- 2016
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15. Conformational activation of visual rhodopsin in native disc membranes
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Malmerberg, E., Bovee-Geurts, P.H.M., Katona, G., Deupi, X., Arnlund, D., Wickstrand, C., Johansson, L.C., Westenhoff, S., Nazarenko, E., GF, X.S., Menzel, A., Grip, W.J. de, Neutze, R., Malmerberg, E., Bovee-Geurts, P.H.M., Katona, G., Deupi, X., Arnlund, D., Wickstrand, C., Johansson, L.C., Westenhoff, S., Nazarenko, E., GF, X.S., Menzel, A., Grip, W.J. de, and Neutze, R.
- Abstract
Contains fulltext : 154870.pdf (publisher's version ) (Closed access), Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested.
- Published
- 2015
16. Night blindness causing G90D rhodopsin in complex with GaCT2 peptide
- Author
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Singhal, A., primary, Ostermaier, M.K., additional, Vishnivetskiy, S.A., additional, Panneels, V., additional, Homan, K.T., additional, Tesmer, J.J.G., additional, Veprintsev, D., additional, Deupi, X., additional, Gurevich, V.V., additional, Schertler, G.F.X., additional, and Standfuss, J., additional
- Published
- 2013
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17. Night blindness causing G90D rhodopsin in the active conformation
- Author
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Singhal, A., primary, Ostermaier, M.K., additional, Vishnivetskiy, S.A., additional, Panneels, V., additional, Homan, K.T., additional, Tesmer, J.J.G., additional, Veprintsev, D., additional, Deupi, X., additional, Gurevich, V.V., additional, Schertler, G.F.X., additional, and Standfuss, J., additional
- Published
- 2013
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18. Crystal structure of the light-activated constitutively active N2C, M257Y,D282C rhodopsin mutant in complex with a peptide resembling the C-terminus of the Galpha-protein subunit (GaCT)
- Author
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Deupi, X., primary, Edwards, P., additional, Singhal, A., additional, Nickle, B., additional, Oprian, D.D., additional, Schertler, G.F.X., additional, and Standfuss, J., additional
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- 2012
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19. The TXP motif in the second transmembrane helix of CCR5. A structural determinant of chemokine-induced activation.
- Author
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Govaerts, C, Blanpain, C, Deupi, X, Ballet, S, Ballesteros, J A, Wodak, S J, Vassart, G, Pardo, L, and Parmentier, M
- Abstract
CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein 1alpha and 1beta, and monocyte chemotactic protein 2 and is the main co-receptor for the macrophage-tropic human immunodeficiency virus strains. We have identified a sequence motif (TXP) in the second transmembrane helix of chemokine receptors and investigated its role by theoretical and experimental approaches. Molecular dynamics simulations of model alpha-helices in a nonpolar environment were used to show that a TXP motif strongly bends these helices, due to the coordinated action of the proline, which kinks the helix, and of the threonine, which further accentuates this structural deformation. Site-directed mutagenesis of the corresponding Pro and Thr residues in CCR5 allowed us to probe the consequences of these structural findings in the context of the whole receptor. The P84A mutation leads to a decreased binding affinity for chemokines and nearly abolishes the functional response of the receptor. In contrast, mutation of Thr-82(2.56) into Val, Ala, Cys, or Ser does not affect chemokine binding. However, the functional response was found to depend strongly on the nature of the substituted side chain. The rank order of impairment of receptor activation is P84A > T82V > T82A > T82C > T82S. This ranking of impairment parallels the bending of the alpha-helix observed in the molecular simulation study.
- Published
- 2001
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20. Development of radiofluorinated MLN-4760 derivatives for PET imaging of the SARS-CoV-2 entry receptor ACE2.
- Author
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Wang J, Beyer D, Vaccarin C, He Y, Tanriver M, Benoit R, Deupi X, Mu L, Bode JW, Schibli R, and Müller C
- Subjects
- Humans, Animals, Mice, HEK293 Cells, Positron-Emission Tomography methods, COVID-19 diagnostic imaging, Radiopharmaceuticals pharmacokinetics, Radiopharmaceuticals chemistry, Radiopharmaceuticals chemical synthesis, Tissue Distribution, Angiotensin-Converting Enzyme 2 metabolism, Fluorine Radioisotopes chemistry, SARS-CoV-2
- Abstract
Purpose: The angiotensin converting enzyme 2 (ACE2) plays a regulatory role in the cardiovascular system and serves SARS-CoV-2 as an entry receptor. The aim of this study was to synthesize and evaluate radiofluorinated derivatives of the ACE2 inhibitor MLN-4760. [
18 F]F-MLN-4760 and [18 F]F-Aza-MLN-4760 were demonstrated to be suitable for non-invasive imaging of ACE2, potentially enabling a better understanding of its expression dynamics., Methods: Computational molecular modeling, based on the structures of human ACE2 (hACE2) and mouse ACE2 (mACE2), revealed that the ACE2-binding modes of F-MLN-4760 and F-Aza-MLN-4760 were similar to that of MLN-4760. Co-crystallization of the hACE2/F-MLN-4760 protein complex was performed for confirmation. Displacement experiments using [3 H]MLN-4760 enabled the determination of the binding affinities of the synthesized F-MLN-4760 and F-Aza-MLN-4760 to hACE2 expressed in HEK-ACE2 cells. Aryl trimethylstannane-based and pyridine-based radiofluorination precursors were synthesized and used for the preparation of the respective radiotracers. [18 F]F-MLN-4760 and [18 F]F-Aza-MLN-4760 were evaluated with regard to the uptake in HEK-ACE2 and HEK-ACE cells and in vitro binding to tissue sections of HEK-ACE2 xenografts and normal organs of mice. Biodistribution and PET/CT imaging studies of [18 F]F-MLN-4760 and [18 F]F-Aza-MLN-4760 were performed using HEK-ACE2 and HEK-ACE xenografted nude mice., Results: Crystallography data revealed an equal hACE2-binding mode for F-MLN-4760 as previously found for MLN-4760. Moreover, computer-based modeling indicated that similar binding to hACE2 and mACE2 holds true for both, F-MLN-4760 and F-Aza-MLN-4760, as is the case for MLN-4760. The IC50 values were three-fold and seven-fold higher for F-MLN-4760 and F-Aza-MLN-4760, respectively, than for MLN-4760. [18 F]F-MLN-4760 and [18 F]F-Aza-MLN-4760 were obtained in 1.4 ± 0.3 GBq and 0.5 ± 0.1 GBq activity with > 99% radiochemical purity in a 5.3% and 1.2% radiochemical yield, respectively. Uptake in HEK-ACE2 cells was higher for [18 F]F-MLN-4760 (67 ± 9%) than for [18 F]F-Aza-MLN-4760 (37 ± 8%) after 3-h incubation while negligible uptake was seen in HEK-ACE cells (< 0.3%). [18 F]F-MLN-4760 and [18 F]F-Aza-MLN-4760 accumulated specifically in HEK-ACE2 xenografts of mice (13 ± 2% IA/g and 15 ± 2% IA/g at 1 h p.i.) with almost no uptake observed in HEK-ACE xenografts (< 0.3% IA/g). This was confirmed by PET/CT imaging, which also visualized unspecific accumulation in the gall bladder and intestinal tract., Conclusion: Both radiotracers showed specific and selective binding to ACE2 in vitro and in vivo. [18 F]F-MLN-4760 was, however, obtained in higher yields and the ACE2-binding affinity was superior over that of [18 F]F-Aza-MLN-4760. [18 F]F-MLN-4760 would, thus, be the candidate of choice for further development in view of its use for PET imaging of ACE2., Competing Interests: Declarations. Ethical approval: This study was performed in agreement with the national law and PSI-internal guidelines of radiation safety protection. In vivo experiments were approved by the local veterinarian department and ethics committee and conducted in accordance with the Swiss law of animal protection. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose. Institutional review board statement: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. In particular, all animal experiments were carried out according to the guidelines of the Swiss Regulations for Animal Welfare. The preclinical studies have been ethically approved by the Cantonal Committee of Animal Experimentation and permitted by the responsible cantonal authorities (License N° 75743)., (© 2024. The Author(s).)- Published
- 2024
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21. Calcineurin-fusion facilitates cryo-EM structure determination of a Family A GPCR.
- Author
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Xu J, Chen G, Wang H, Cao S, Heng J, Deupi X, Du Y, and Kobilka BK
- Subjects
- Humans, Receptors, G-Protein-Coupled metabolism, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled ultrastructure, Models, Molecular, Recombinant Fusion Proteins metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins ultrastructure, Protein Conformation, HEK293 Cells, Protein Domains, Cryoelectron Microscopy methods, Calcineurin metabolism, Calcineurin chemistry, Receptors, Adrenergic, beta-2 metabolism, Receptors, Adrenergic, beta-2 chemistry, Receptors, Adrenergic, beta-2 ultrastructure
- Abstract
Advances in singe-particle cryo-electron microscopy (cryo-EM) have made it possible to solve the structures of numerous Family A and Family B G protein-coupled receptors (GPCRs) in complex with G proteins and arrestins, as well as several Family C GPCRs. Determination of these structures has been facilitated by the presence of large extramembrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during the processing of the cryo-EM data. In contrast, determination of the inactive state structure of Family A GPCRs is more challenging due to the relatively small size of the seven transmembrane domain (7TM) and to the surrounding detergent micelle that, in the absence of other features, make particle alignment impossible. Here, we describe an alternative protein engineering strategy where the heterodimeric protein calcineurin is fused to a GPCR by three points of attachment, the cytoplasmic ends of TM5, TM6, and TM7. This three-point attachment provides a more rigid link with the GPCR transmembrane domain that facilitates particle alignment during data processing, allowing us to determine the structures of the β
2 adrenergic receptor (β2 AR) in the apo, antagonist-bound, and agonist-bound states. We expect that this fusion strategy may have broad application in cryo-EM structural determination of other Family A GPCRs., Competing Interests: Competing interests statement:Brian Kobilka is a co-founder of an consultant for ConfometRx. Confometrx has no financial interest in this paper. Brian Kobilka is a co-founder of ConfmetRx.- Published
- 2024
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22. Active state structures of a bistable visual opsin bound to G proteins.
- Author
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Tejero O, Pamula F, Koyanagi M, Nagata T, Afanasyev P, Das I, Deupi X, Sheves M, Terakita A, Schertler GFX, Rodrigues MJ, and Tsai CJ
- Subjects
- Animals, Rhodopsin metabolism, Rhodopsin chemistry, Humans, Protein Binding, Spiders metabolism, Signal Transduction, Models, Molecular, Crystallography, X-Ray, GTP-Binding Proteins metabolism, GTP-Binding Proteins chemistry, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, GTP-Binding Protein alpha Subunits, Gq-G11 chemistry, Opsins metabolism, Opsins chemistry, Opsins genetics
- Abstract
Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the G
i and Gq heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways., (© 2024. The Author(s).)- Published
- 2024
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23. Study of Photoselectivity in Linear Conjugated Chromophores Using the XMS-CASPT2 Method.
- Author
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Sen S and Deupi X
- Abstract
Photoisomerization, the structural alteration of molecules upon absorption of light, is crucial for the function of biological chromophores such as retinal in opsins, proteins vital for vision and other light-sensitive processes. The intrinsic selectivity of this isomerization process (i.e., which double bond in the chromophore is isomerized) is governed by both the inherent properties of the chromophore and its surrounding environment. In this study, we employ the extended multistate complete active space second-order perturbation theory (XMS-CASPT2) method to investigate photoisomerization selectivity in linear conjugated chromophores, focusing on two simple molecular models resembling retinal. By analyzing electronic energies, intramolecular charge separation, and conical intersection topographies in the gas phase, we show that the photoproduct formed by rotation around the double bond near the Schiff base is energetically favored. The topographic differences at the conical intersections leading to different photoproducts reveal differences in photodynamics. In multiphoton excitation, the primary photoproduct typically reverts to the initial configuration rather than rotating around a different double bond. Our study offers new insights into the photodynamics of photoisomerizing double bonds in π-conjugated chromophores. We anticipate that our findings will provide valuable perspectives for advancing the understanding of biological chromophores and for designing efficient photochemical switches with applications in molecular electronics and phototherapy., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)
- Published
- 2024
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24. A tool for nuclear imaging of the SARS-CoV-2 entry receptor: molecular model and preclinical development of ACE2-selective radiopeptides.
- Author
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Beyer D, Vaccarin C, Deupi X, Mapanao AK, Cohrs S, Sozzi-Guo F, Grundler PV, van der Meulen NP, Wang J, Tanriver M, Bode JW, Schibli R, and Müller C
- Abstract
Purpose: The angiotensin converting enzyme-2 (ACE2)-entry receptor of SARS-CoV-2-and its homologue, the angiotensin-converting enzyme (ACE), play a pivotal role in maintaining cardiovascular homeostasis. Potential changes in ACE2 expression levels and dynamics after SARS-CoV-2 infection have been barely investigated. The aim of this study was to develop an ACE2-targeting imaging agent as a noninvasive imaging tool to determine ACE2 regulation., Methods: DOTA-DX600, NODAGA-DX600 and HBED-CC-DX600 were obtained through custom synthesis and labeled with gallium-67 (T
1/2 = 3.26 d) as a surrogate radioisotope for gallium-68 (T1/2 = 68 min). ACE2- and ACE-transfected HEK cells were used for the in vitro evaluation of these radiopeptides. The in vivo tissue distribution profiles of the radiopeptides were assessed in HEK-ACE2 and HEK-ACE xenografted mice and imaging studies were performed using SPECT/CT., Results: The highest molar activity was obtained for [67 Ga]Ga-HBED-CC-DX600 (60 MBq/nmol), whereas the labeling efficiency of the other peptides was considerably lower (20 MBq/nmol). The radiopeptides were stable over 24 h in saline (> 99% intact peptide). All radiopeptides showed uptake in HEK-ACE2 cells (36-43%) with moderate ACE2-binding affinity (KD value: 83-113 nM), but no uptake in HEK-ACE cells (< 0.1%) was observed. Accumulation of the radiopeptides was observed in HEK-ACE2 xenografts (11-16% IA/g) at 3 h after injection, but only background signals were seen in HEK-ACE xenografts (< 0.5% IA/g). Renal retention was still high 3 h after injection of [67 Ga]Ga-DOTA-DX600 and [67 Ga]Ga-NODAGA-DX600 (~ 24% IA/g), but much lower for [67 Ga]Ga-HBED-CC-DX600 (7.2 ± 2.2% IA/g). SPECT/CT imaging studies confirmed the most favorable target-to-nontarget ratio for [67 Ga]Ga-HBED-CC-DX600., Conclusions: This study demonstrated ACE2 selectivity for all radiopeptides. [67 Ga]Ga-HBED-CC-DX600 was revealed as the most promising candidate due to its favorable tissue distribution profile. Importantly, the HBED-CC chelator enabled67 Ga-labeling at high molar activity, which would be essential to obtain images with high signal-to-background contrast to detect (patho)physiological ACE2 expression levels in patients., (© 2023. The Author(s).)- Published
- 2023
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25. Ultrafast structural changes direct the first molecular events of vision.
- Author
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Gruhl T, Weinert T, Rodrigues MJ, Milne CJ, Ortolani G, Nass K, Nango E, Sen S, Johnson PJM, Cirelli C, Furrer A, Mous S, Skopintsev P, James D, Dworkowski F, Båth P, Kekilli D, Ozerov D, Tanaka R, Glover H, Bacellar C, Brünle S, Casadei CM, Diethelm AD, Gashi D, Gotthard G, Guixà-González R, Joti Y, Kabanova V, Knopp G, Lesca E, Ma P, Martiel I, Mühle J, Owada S, Pamula F, Sarabi D, Tejero O, Tsai CJ, Varma N, Wach A, Boutet S, Tono K, Nogly P, Deupi X, Iwata S, Neutze R, Standfuss J, Schertler G, and Panneels V
- Subjects
- Animals, Binding Sites radiation effects, Crystallography, Heterotrimeric GTP-Binding Proteins chemistry, Heterotrimeric GTP-Binding Proteins metabolism, Isomerism, Photons, Protein Binding radiation effects, Protein Conformation radiation effects, Retinaldehyde chemistry, Retinaldehyde metabolism, Retinaldehyde radiation effects, Time Factors, Rhodopsin chemistry, Rhodopsin metabolism, Rhodopsin radiation effects, Vision, Ocular physiology, Vision, Ocular radiation effects
- Abstract
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)
1 . A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2 , thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation., (© 2023. The Author(s).)- Published
- 2023
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26. β-arrestin1 and 2 exhibit distinct phosphorylation-dependent conformations when coupling to the same GPCR in living cells.
- Author
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Haider RS, Matthees ESF, Drube J, Reichel M, Zabel U, Inoue A, Chevigné A, Krasel C, Deupi X, and Hoffmann C
- Subjects
- G-Protein-Coupled Receptor Kinases metabolism, Luciferases, Parathyroid Hormone metabolism, Phosphorylation physiology, Protein Isoforms metabolism, Receptors, G-Protein-Coupled metabolism, beta-Arrestin 1 genetics, beta-Arrestin 1 metabolism, beta-Arrestin 2 genetics, beta-Arrestin 2 metabolism, beta-Arrestins metabolism, Arrestins metabolism, Signal Transduction physiology
- Abstract
β-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR-β-arrestin complex. However, whether β-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and β-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations ("hanging" and "core"). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of β-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess β-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function., (© 2022. The Author(s).)
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- 2022
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27. Structural Elements Directing G Proteins and β-Arrestin Interactions with the Human Melatonin Type 2 Receptor Revealed by Natural Variants.
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Plouffe B, Karamitri A, Flock T, Gallion JM, Houston S, Daly CA, Bonnefond A, Guillaume JL, Le Gouill C, Froguel P, Lichtarge O, Deupi X, Jockers R, and Bouvier M
- Abstract
G protein-coupled receptors (GPCRs) can engage distinct subsets of signaling pathways, but the structural determinants of this functional selectivity remain elusive. The naturally occurring genetic variants of GPCRs, selectively affecting different pathways, offer an opportunity to explore this phenomenon. We previously identified 40 coding variants of the MTNR1B gene encoding the melatonin MT
2 receptor (MT2 ). These mutations differently impact the β-arrestin 2 recruitment, ERK activation, cAMP production, and Gαi1 and Gαz activation. In this study, we combined functional clustering and structural modeling to delineate the molecular features controlling the MT2 functional selectivity. Using non-negative matrix factorization, we analyzed the signaling signatures of the 40 MT2 variants yielding eight clusters defined by unique signaling features and localized in distinct domains of MT2 . Using computational homology modeling, we describe how specific mutations can selectively affect the subsets of signaling pathways and offer a proof of principle that natural variants can be used to explore and understand the GPCR functional selectivity., Competing Interests: The authors declare the following competing financial interest(s): M.B. is the president of the scientific advisory Board of Domain Therapeutics which licensed-in some of the BRET-based biosensors, used in the present study, for their commercial use., (© 2022 The Authors. Published by American Chemical Society.)- Published
- 2022
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28. Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology.
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Collu G, Bierig T, Krebs AS, Engilberge S, Varma N, Guixà-González R, Sharpe T, Deupi X, Olieric V, Poghosyan E, and Benoit RM
- Subjects
- Cryoelectron Microscopy, Crystallography, X-Ray, Models, Molecular, Molecular Dynamics Simulation, Nanoparticles, Protein Structure, Secondary, Epitopes chemistry, Recombinant Fusion Proteins chemistry
- Abstract
Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH), can be seamlessly fused to terminal helices of proteins, forming an extended, partially free-standing rigid helix. This enables the connection of two domains at a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano. Analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example, to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact on construct flexibility (for the study of protein dynamics), and to design novel biomaterials., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Ltd.)
- Published
- 2022
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29. Exploring the signaling space of a GPCR using bivalent ligands with a rigid oligoproline backbone.
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Romantini N, Alam S, Dobitz S, Spillmann M, De Foresta M, Schibli R, Schertler GFX, Wennemers H, Deupi X, Behe M, and Berger P
- Subjects
- Allosteric Site, Amino Acid Motifs, Cloning, Molecular, Dimerization, HEK293 Cells, Humans, Kinetics, Ligands, Peptides chemistry, Protein Engineering methods, Signal Transduction, beta-Arrestins metabolism, Proline chemistry, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled metabolism
- Abstract
G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward β-arrestin-1 and β-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting β-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)
- Published
- 2021
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30. Unraveling binding mechanism and kinetics of macrocyclic Gα q protein inhibitors.
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Voss JH, Nagel J, Rafehi M, Guixà-González R, Malfacini D, Patt J, Kehraus S, Inoue A, König GM, Kostenis E, Deupi X, Namasivayam V, and Müller CE
- Subjects
- GTP-Binding Protein alpha Subunits, Gq-G11 chemistry, GTP-Binding Protein alpha Subunits, Gq-G11 genetics, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, HEK293 Cells, Humans, Kinetics, Models, Molecular, Protein Binding, Depsipeptides pharmacology, GTP-Binding Protein alpha Subunits, Gq-G11 antagonists & inhibitors, Peptides, Cyclic pharmacology
- Abstract
G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gα
q protein family. We recently discovered that radiolabeled FR and YM display strongly divergent residence times, which translates into significantly longer antiasthmatic effects of FR. The present study is aimed at investigating the molecular basis for this observed disparity. Based on docking studies, we mutated amino acid residues of the Gαq protein predicted to interact with FR or YM, and recombinantly expressed the mutated Gαq proteins in cells in which the native Gαq proteins had been knocked out by CRISPR-Cas9. Both radioligands showed similar association kinetics, and their binding followed a conformational selection mechanism, which was rationalized by molecular dynamics simulation studies. Several mutations of amino acid residues near the putative binding site of the "lipophilic anchors" of FR, especially those predicted to interact with the isopropyl group present in FR but not in YM, led to dramatically accelerated dissociation kinetics. Our data indicate that the long residence time of FR depends on lipophilic interactions within its binding site. The observed structure-kinetic relationships point to a complex binding mechanism of FR, which likely involves snap-lock- or dowel-like conformational changes of either ligand or protein, or both. These experimental data will be useful for the design of compounds with a desired residence time, a parameter that has now been recognized to be of utmost importance in drug development., (Copyright © 2021. Published by Elsevier Ltd.)- Published
- 2021
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31. Identification of Key Regions Mediating Human Melatonin Type 1 Receptor Functional Selectivity Revealed by Natural Variants.
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Hegron A, Huh E, Deupi X, Sokrat B, Gao W, Le Gouill C, Canouil M, Boissel M, Charpentier G, Roussel R, Balkau B, Froguel P, Plouffe B, Bonnefond A, Lichtarge O, Jockers R, and Bouvier M
- Abstract
Melatonin is a hormone mainly produced by the pineal gland and MT
1 is one of the two G protein-coupled receptors (GPCRs) mediating its action. Despite an increasing number of available GPCR crystal structures, the molecular mechanism of activation of a large number of receptors, including MT1 , remains poorly understood. The purpose of this study is to elucidate the structural elements involved in the process of MT1 's activation using naturally occurring variants affecting its function. Thirty-six nonsynonymous variants, including 34 rare ones, were identified in MTNR1A (encoding MT1 ) from a cohort of 8687 individuals and their signaling profiles were characterized using Bioluminescence Resonance Energy Transfer-based sensors probing 11 different signaling pathways. Computational analysis of the experimental data allowed us to group the variants in clusters according to their signaling profiles and to analyze the position of each variant in the context of the three-dimensional structure of MT1 to link functional selectivity to structure. MT1 variant signaling profiles revealed three clusters characterized by (1) wild-type-like variants, (2) variants with selective defect of βarrestin-2 recruitment, and (3) severely defective variants on all pathways. Our structural analysis allows us to identify important regions for βarrestin-2 recruitment as well as for Gα12 and Gα15 activation. In addition to identifying MT1 domains differentially controlling the activation of the various signaling effectors, this study illustrates how natural variants can be used as tools to study the molecular mechanisms of receptor activation., Competing Interests: The authors declare the following competing financial interest(s): M.B. is the president of the scientific advisory board of Domain Therapeutics which licenced-in some of the BRET-based biosensors used in the present study for their commercial use. All other authors declare no competing interests., (© 2021 The Authors. Published by American Chemical Society.)- Published
- 2021
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32. High-mass MALDI-MS unravels ligand-mediated G protein-coupling selectivity to GPCRs.
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Wu N, Olechwier AM, Brunner C, Edwards PC, Tsai CJ, Tate CG, Schertler GFX, Schneider G, Deupi X, Zenobi R, and Ma P
- Subjects
- Animals, Arrestin genetics, Arrestin metabolism, GTP-Binding Proteins genetics, Gene Expression Regulation, HEK293 Cells, Humans, Ligands, Mice, Models, Molecular, Protein Binding, Protein Conformation, Receptors, Opioid chemistry, Single-Chain Antibodies, Turkeys, beta-Arrestin 1 genetics, beta-Arrestin 1 metabolism, GTP-Binding Proteins metabolism, Receptors, Opioid metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
G protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR-G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand-GPCR-partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [β1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using β1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gα
i βγ and β-arrestin-1 and showed that carvedilol induces an increase in coupling of β-arrestin-1 and Gαi βγ to β1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs., Competing Interests: Competing interest statement: C.G.T. is a shareholder, consultant, and member of the scientific advisory board of Sosei Heptares. G.F.X.S. declares that he is a cofounder and scientific advisor of the companies leadXpro AG and InterAx Biotech AG. G.S. is a cofounder of inSili.com LLC and a consultant to the pharmaceutical industry. N.W., A.M.O., G.F.X.S., X.D., R.Z., and P.M. are inventors on a patent application filed by the Paul Scherrer Institute relating to the content in this paper., (Copyright © 2021 the Author(s). Published by PNAS.)- Published
- 2021
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33. Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist.
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Isaikina P, Tsai CJ, Dietz N, Pamula F, Grahl A, Goldie KN, Guixà-González R, Branco C, Paolini-Bertrand M, Calo N, Cerini F, Schertler GFX, Hartley O, Stahlberg H, Maier T, Deupi X, and Grzesiek S
- Subjects
- Chemokine CCL5 chemistry, Chemokine CCL5 metabolism, Cryoelectron Microscopy, Humans, Models, Molecular, Molecular Dynamics Simulation, Protein Conformation, Receptors, CCR5 agonists, Receptors, CCR5 genetics, Signal Transduction, Structure-Activity Relationship, Receptors, CCR5 chemistry, Receptors, CCR5 metabolism
- Abstract
The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric G
i protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)- Published
- 2021
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34. An experimental strategy to probe Gq contribution to signal transduction in living cells.
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Patt J, Alenfelder J, Pfeil EM, Voss JH, Merten N, Eryilmaz F, Heycke N, Rick U, Inoue A, Kehraus S, Deupi X, Müller CE, König GM, Crüsemann M, and Kostenis E
- Subjects
- Depsipeptides pharmacology, GTP-Binding Protein alpha Subunits metabolism, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, HEK293 Cells, Heterotrimeric GTP-Binding Proteins metabolism, Heterotrimeric GTP-Binding Proteins physiology, Humans, Peptides, Cyclic pharmacology, Signal Transduction drug effects, GTP-Binding Protein alpha Subunits physiology, GTP-Binding Protein alpha Subunits, Gq-G11 physiology, Signal Transduction physiology
- Abstract
Heterotrimeric G protein subunits Gαq and Gα11 are inhibited by two cyclic depsipeptides, FR900359 (FR) and YM-254890 (YM), both of which are being used widely to implicate Gq/11 proteins in the regulation of diverse biological processes. An emerging major research question therefore is whether the cellular effects of both inhibitors are on-target, that is, mediated via specific inhibition of Gq/11 proteins, or off-target, that is, the result of nonspecific interactions with other proteins. Here we introduce a versatile experimental strategy to discriminate between these possibilities. We developed a Gαq variant with preserved catalytic activity, but refractory to FR/YM inhibition. A minimum of two amino acid changes were required and sufficient to achieve complete inhibitor resistance. We characterized the novel mutant in HEK293 cells depleted by CRISPR-Cas9 of endogenous Gαq and Gα11 to ensure precise control over the Gα-dependent cellular signaling route. Using a battery of cellular outcomes with known and concealed Gq contribution, we found that FR/YM specifically inhibited cellular signals after Gαq introduction via transient transfection. Conversely, both inhibitors were inert across all assays in cells expressing the drug-resistant variant. These findings eliminate the possibility that inhibition of non-Gq proteins contributes to the cellular effects of the two depsipeptides. We conclude that combined application of FR or YM along with the drug-resistant Gαq variant is a powerful in vitro strategy to discern on-target Gq against off-target non-Gq action. Consequently, it should be of high value for uncovering Gq input to complex biological processes with high accuracy and the requisite specificity., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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35. Distance-Dependent Cellular Uptake of Oligoproline-Based Homobivalent Ligands Targeting GPCRs-An Experimental and Computational Analysis.
- Author
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Dobitz S, Wilhelm P, Romantini N, De Foresta M, Walther C, Ritler A, Schibli R, Berger P, Deupi X, Béhé M, and Wennemers H
- Subjects
- Animals, Cell Line, Tumor, Female, HEK293 Cells, Humans, Ligands, Mice, SCID, Molecular Dynamics Simulation, Neoplasms metabolism, Oligopeptides pharmacology, Proline pharmacology, Receptors, Bombesin metabolism, Receptors, Somatostatin metabolism, Oligopeptides chemistry, Oligopeptides pharmacokinetics, Proline analogs & derivatives, Proline pharmacokinetics, Receptors, Bombesin agonists, Receptors, Somatostatin agonists
- Abstract
Tumor targeting with bivalent radiolabeled ligands for GPCRs is an attractive means for cancer imaging and therapy. Here, we studied and compared the distance dependence of homobivalent ligands for the human gastrin-releasing peptide receptor (hGRP-R) and the somatostatin receptor subtype II (hSstR
2a ). Oligoprolines were utilized as molecular scaffolds to enable distances of 10, 20, or 30 Å between two identical, agonistic recognition motifs. In vitro internalization assays revealed that ligands with a distance of 20 Å between the recognition motifs exhibit the highest cellular uptake in both ligand series. Structural modeling and molecular dynamics simulations support an optimal distance of 20 Å for accommodating ligand binding to both binding sites of a GPCR dimer. Translation of these findings to the significantly higher complexity in vivo proved difficult and showed only for the hGRP-R increased tumor uptake of the bivalent ligand.- Published
- 2020
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36. GPCRmd uncovers the dynamics of the 3D-GPCRome.
- Author
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Rodríguez-Espigares I, Torrens-Fontanals M, Tiemann JKS, Aranda-García D, Ramírez-Anguita JM, Stepniewski TM, Worp N, Varela-Rial A, Morales-Pastor A, Medel-Lacruz B, Pándy-Szekeres G, Mayol E, Giorgino T, Carlsson J, Deupi X, Filipek S, Filizola M, Gómez-Tamayo JC, Gonzalez A, Gutiérrez-de-Terán H, Jiménez-Rosés M, Jespers W, Kapla J, Khelashvili G, Kolb P, Latek D, Marti-Solano M, Matricon P, Matsoukas MT, Miszta P, Olivella M, Perez-Benito L, Provasi D, Ríos S, R Torrecillas I, Sallander J, Sztyler A, Vasile S, Weinstein H, Zachariae U, Hildebrand PW, De Fabritiis G, Sanz F, Gloriam DE, Cordomi A, Guixà-González R, and Selent J
- Subjects
- Metabolome, Models, Molecular, Protein Conformation, Molecular Dynamics Simulation, Receptors, G-Protein-Coupled chemistry, Software
- Abstract
G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.
- Published
- 2020
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37. Publisher Correction: GPCRmd uncovers the dynamics of the 3D-GPCRome.
- Author
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Rodríguez-Espigares I, Torrens-Fontanals M, Tiemann JKS, Aranda-García D, Ramírez-Anguita JM, Stepniewski TM, Worp N, Varela-Rial A, Morales-Pastor A, Medel-Lacruz B, Pándy-Szekeres G, Mayol E, Giorgino T, Carlsson J, Deupi X, Filipek S, Filizola M, Gómez-Tamayo JC, Gonzalez A, Gutiérrez-de-Terán H, Jiménez-Rosés M, Jespers W, Kapla J, Khelashvili G, Kolb P, Latek D, Marti-Solano M, Matricon P, Matsoukas MT, Miszta P, Olivella M, Perez-Benito L, Provasi D, Ríos S, R Torrecillas I, Sallander J, Sztyler A, Vasile S, Weinstein H, Zachariae U, Hildebrand PW, De Fabritiis G, Sanz F, Gloriam DE, Cordomi A, Guixà-González R, and Selent J
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2020
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38. Femtosecond-to-millisecond structural changes in a light-driven sodium pump.
- Author
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Skopintsev P, Ehrenberg D, Weinert T, James D, Kar RK, Johnson PJM, Ozerov D, Furrer A, Martiel I, Dworkowski F, Nass K, Knopp G, Cirelli C, Arrell C, Gashi D, Mous S, Wranik M, Gruhl T, Kekilli D, Brünle S, Deupi X, Schertler GFX, Benoit RM, Panneels V, Nogly P, Schapiro I, Milne C, Heberle J, and Standfuss J
- Subjects
- Binding Sites, Crystallography, Electrons, Ion Transport, Isomerism, Lasers, Protons, Quantum Theory, Retinaldehyde chemistry, Retinaldehyde metabolism, Schiff Bases chemistry, Sodium metabolism, Spectrum Analysis, Static Electricity, Time Factors, Flavobacteriaceae chemistry, Rhodopsins, Microbial chemistry, Rhodopsins, Microbial radiation effects, Sodium-Potassium-Exchanging ATPase chemistry, Sodium-Potassium-Exchanging ATPase radiation effects
- Abstract
Light-driven sodium pumps actively transport small cations across cellular membranes
1 . These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3 , it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4 , we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.- Published
- 2020
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39. Triazolo-Peptidomimetics: Novel Radiolabeled Minigastrin Analogs for Improved Tumor Targeting.
- Author
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Grob NM, Häussinger D, Deupi X, Schibli R, Behe M, and Mindt TL
- Subjects
- Animals, Antineoplastic Agents chemical synthesis, Antineoplastic Agents metabolism, Antineoplastic Agents pharmacokinetics, Cell Line, Tumor, Female, Gastrins chemical synthesis, Gastrins metabolism, Gastrins pharmacokinetics, Humans, Lutetium chemistry, Mice, Neoplasms metabolism, Peptidomimetics chemical synthesis, Peptidomimetics metabolism, Peptidomimetics pharmacokinetics, Protein Binding, Protein Conformation, Radioisotopes chemistry, Radiopharmaceuticals chemical synthesis, Radiopharmaceuticals metabolism, Radiopharmaceuticals pharmacokinetics, Receptor, Cholecystokinin B metabolism, Triazoles chemical synthesis, Triazoles metabolism, Triazoles pharmacokinetics, Antineoplastic Agents pharmacology, Gastrins pharmacology, Peptidomimetics pharmacology, Radiopharmaceuticals pharmacology, Triazoles pharmacology
- Abstract
MG11 is a truncated analog of minigastrin, a peptide with high affinity and specificity toward the cholecystokinin-2 receptor (CCK2R), which is overexpressed by different tumors. Thus, radiolabeled MG11 derivatives have great potential for use in cancer diagnosis and therapy. A drawback of MG11 is its fast degradation by proteases, leading to moderate tumor uptake in vivo . We introduced 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres to replace labile amide bonds of the peptide. The "triazole scan" yielded peptidomimetics with improved resistance to enzymatic degradation and/or enhanced affinity toward the CCK2R. Remarkably, our lead compound achieved a 10-fold increase in receptor affinity, resulting in a 2.6-fold improved tumor uptake in vivo . Modeling of the ligand-CCK2R complex suggests that an additional cation-π interaction of the aromatic triazole moiety with the Arg
356 residue of the receptor is accountable for these observations. We show for the first time that the amide-to-triazole substitution strategy offers new opportunities in drug development that go beyond the metabolic stabilization of bioactive peptides.- Published
- 2020
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40. Crystal structure of jumping spider rhodopsin-1 as a light sensitive GPCR.
- Author
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Varma N, Mutt E, Mühle J, Panneels V, Terakita A, Deupi X, Nogly P, Schertler GFX, and Lesca E
- Subjects
- Animals, Arthropod Proteins isolation & purification, Arthropod Proteins metabolism, Crystallography, X-Ray, HEK293 Cells, Humans, Ligands, Light, Molecular Dynamics Simulation, Protein Isoforms isolation & purification, Protein Isoforms metabolism, Protein Isoforms ultrastructure, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Recombinant Proteins ultrastructure, Rhodopsin isolation & purification, Rhodopsin metabolism, Stereoisomerism, Structure-Activity Relationship, Arthropod Proteins ultrastructure, Rhodopsin ultrastructure, Signal Transduction radiation effects, Spiders
- Abstract
Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9- cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs., Competing Interests: Conflict of interest statement: G.F.X.S. declares that he is a cofounder and scientific advisor of the companies leadXpro AG and InterAx Biotech AG., (Copyright © 2019 the Author(s). Published by PNAS.)
- Published
- 2019
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41. Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the gβ subunit.
- Author
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Tsai CJ, Marino J, Adaixo R, Pamula F, Muehle J, Maeda S, Flock T, Taylor NM, Mohammed I, Matile H, Dawson RJ, Deupi X, Stahlberg H, and Schertler G
- Subjects
- Animals, Cattle, Cryoelectron Microscopy, GTP-Binding Protein beta Subunits metabolism, Multiprotein Complexes ultrastructure, Protein Binding, Rhodopsin metabolism, GTP-Binding Protein alpha Subunits ultrastructure, GTP-Binding Protein beta Subunits ultrastructure, GTP-Binding Protein gamma Subunits ultrastructure, Rhodopsin ultrastructure
- Abstract
One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway., Competing Interests: CT, JM, RA, FP, JM, SM, TF, NT, IM, XD, HS No competing interests declared, HM, RD Employee of Hoffmann-La Roche Ltd, GS declares that he is a co-founder and scientific advisor of the company leadXpro AG and InterAx Biotech AG, and that he has been a member of the MAX IV Scientific Advisory Committee during the time when the research has been performed., (© 2019, Tsai et al.)
- Published
- 2019
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42. The counterion-retinylidene Schiff base interaction of an invertebrate rhodopsin rearranges upon light activation.
- Author
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Nagata T, Koyanagi M, Tsukamoto H, Mutt E, Schertler GFX, Deupi X, and Terakita A
- Subjects
- Amino Acid Substitution, Animals, Arthropod Proteins genetics, Hydrogen Bonding, Light, Models, Molecular, Mutagenesis, Site-Directed, Photochemical Processes, Protein Stability, Rhodopsin genetics, Schiff Bases chemistry, Schiff Bases radiation effects, Spiders chemistry, Spiders genetics, Arthropod Proteins chemistry, Arthropod Proteins radiation effects, Rhodopsin chemistry, Rhodopsin radiation effects
- Abstract
Animals sense light using photosensitive proteins-rhodopsins-containing a chromophore-retinal-that intrinsically absorbs in the ultraviolet. Visible light-sensitivity depends primarily on protonation of the retinylidene Schiff base (SB), which requires a negatively-charged amino acid residue-counterion-for stabilization. Little is known about how the most common counterion among varied rhodopsins, Glu181, functions. Here, we demonstrate that in a spider visual rhodopsin, orthologue of mammal melanopsins relevant to circadian rhythms, the Glu181 counterion functions likely by forming a hydrogen-bonding network, where Ser186 is a key mediator of the Glu181-SB interaction. We also suggest that upon light activation, the Glu181-SB interaction rearranges while Ser186 changes its contribution. This is in contrast to how the counterion of vertebrate visual rhodopsins, Glu113, functions, which forms a salt bridge with the SB. Our results shed light on the molecular mechanisms of visible light-sensitivity relevant to invertebrate vision and vertebrate non-visual photoreception., Competing Interests: The authors declare no competing interests.
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- 2019
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43. The Two-Photon Reversible Reaction of the Bistable Jumping Spider Rhodopsin-1.
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Ehrenberg D, Varma N, Deupi X, Koyanagi M, Terakita A, Schertler GFX, Heberle J, and Lesca E
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- Absorption, Radiation, Animals, Insect Proteins radiation effects, Protein Domains, Rhodopsin radiation effects, Schiff Bases chemistry, Spiders, Ultraviolet Rays, Insect Proteins chemistry, Photons, Rhodopsin chemistry
- Abstract
Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore-attached to the protein through a protonated Schiff base-and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
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- 2019
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44. Elucidating the Structure-Activity Relationship of the Pentaglutamic Acid Sequence of Minigastrin with Cholecystokinin Receptor Subtype 2.
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Ritler A, Shoshan MS, Deupi X, Wilhelm P, Schibli R, Wennemers H, and Béhé M
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Tumor, Drug Stability, Gastrins pharmacokinetics, Humans, Mice, Molecular Docking Simulation, Structure-Activity Relationship, Tissue Distribution, Gastrins chemistry, Gastrins pharmacology, Receptor, Cholecystokinin B metabolism
- Abstract
Derivatized minigastrin analogues make up a promising class of candidates for targeting cholecystokinin receptor subtype 2 (CCK2R), which is overexpressed on cancer cells of various neuroendocrine tumors. The pentaglutamic acid sequence of minigastrin influences its biological properties. In particular, it plays a crucial role in the kidney reuptake mechanism. However, the importance of the binding affinity and interaction of this region with the receptor on a molecular level remains unclear. To elucidate its structure-activity relationship with CCK2R, we replaced this sequence with various linkers differing in their amount of anionic charge, structural characteristics, and flexibility. Specifically, a flexible aliphatic linker, a linker with only three d-Glu residues, and a structured linker with four adjacent β
3 -glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[d-Glu1-6 ,Nle11 ]gastrin-13). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the minigastrin derivatives, which allowed radiolabeling with Lutetium-177. The levels of In vitro internalization into MZ-CRC1 cells and in vivo tumor uptake as well as human blood plasma stability increased in the following order: aliphatic linker < three d-Glu < (β3 -Glu)4 < (d-Glu)6 . The in vitro and in vivo behavior was therefore significantly improved with anionic charges. Computational modeling of a CCK2 receptor-ligand complex revealed ionic interactions between cationic residues (Arg and His) of the receptor and anionic residues of the ligand in the linker.- Published
- 2019
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45. Distinct G protein-coupled receptor phosphorylation motifs modulate arrestin affinity and activation and global conformation.
- Author
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Mayer D, Damberger FF, Samarasimhareddy M, Feldmueller M, Vuckovic Z, Flock T, Bauer B, Mutt E, Zosel F, Allain FHT, Standfuss J, Schertler GFX, Deupi X, Sommer ME, Hurevich M, Friedler A, and Veprintsev DB
- Subjects
- Amino Acid Motifs physiology, Animals, Arrestin chemistry, Arrestin genetics, Arrestin isolation & purification, Biological Assay, Cattle, Cell Membrane metabolism, Mutation, Nuclear Magnetic Resonance, Biomolecular, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Rhodopsin chemistry, Rod Cell Outer Segment metabolism, beta-Arrestin 1 chemistry, beta-Arrestin 1 isolation & purification, beta-Arrestin 2 chemistry, beta-Arrestin 2 isolation & purification, Arrestin metabolism, Phosphorylation physiology, Rhodopsin metabolism, beta-Arrestin 1 metabolism, beta-Arrestin 2 metabolism
- Abstract
Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: 'key sites' required for arrestin binding and activation, an 'inhibitory site' that abrogates arrestin binding, and 'modulator sites' that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.
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- 2019
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46. An online resource for GPCR structure determination and analysis.
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Munk C, Mutt E, Isberg V, Nikolajsen LF, Bibbe JM, Flock T, Hanson MA, Stevens RC, Deupi X, and Gloriam DE
- Subjects
- Allosteric Site, Animals, Cattle, Cryoelectron Microscopy, Crystallography, X-Ray, Databases, Protein, Drug Design, Glycosylation, HEK293 Cells, Humans, Mutation, Phosphorylation, Protein Domains, Protein Engineering, Rhodopsin chemistry, Signal Transduction, Software, Computational Biology methods, Internet, Receptors, G-Protein-Coupled genetics
- Abstract
G-protein-coupled receptors (GPCRs) transduce physiological and sensory stimuli into appropriate cellular responses and mediate the actions of one-third of drugs. GPCR structural studies have revealed the general bases of receptor activation, signaling, drug action and allosteric modulation, but so far cover only 13% of nonolfactory receptors. We broadly surveyed the receptor modifications/engineering and methods used to produce all available GPCR crystal and cryo-electron microscopy (cryo-EM) structures, and present an interactive resource integrated in GPCRdb ( http://www.gpcrdb.org ) to assist users in designing constructs and browsing appropriate experimental conditions for structure studies.
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- 2019
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47. Arrestin-1 engineering facilitates complex stabilization with native rhodopsin.
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Haider RS, Wilhelm F, Rizk A, Mutt E, Deupi X, Peterhans C, Mühle J, Berger P, Schertler GFX, Standfuss J, and Ostermaier MK
- Subjects
- Animals, Arrestins chemistry, Arrestins genetics, Cattle, HEK293 Cells, Humans, Models, Molecular, Multiprotein Complexes chemistry, Multiprotein Complexes genetics, Mutation, Opsins chemistry, Phosphorylation, Protein Binding, Protein Conformation, Protein Stability, Rhodopsin chemistry, Arrestins metabolism, Multiprotein Complexes metabolism, Opsins metabolism, Protein Engineering methods, Rhodopsin metabolism
- Abstract
Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin-arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC
50 ) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin-arrestin-1 complex. The achieved stabilization of the active rhodopsin-arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).- Published
- 2019
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48. Crystal structure of rhodopsin in complex with a mini-G o sheds light on the principles of G protein selectivity.
- Author
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Tsai CJ, Pamula F, Nehmé R, Mühle J, Weinert T, Flock T, Nogly P, Edwards PC, Carpenter B, Gruhl T, Ma P, Deupi X, Standfuss J, Tate CG, and Schertler GFX
- Subjects
- Animals, Binding Sites, Cattle, Crystallography, X-Ray, Models, Molecular, Multiprotein Complexes chemistry, Multiprotein Complexes metabolism, Mutation, Protein Conformation, Receptors, G-Protein-Coupled metabolism, Rhodopsin genetics, Receptors, G-Protein-Coupled chemistry, Rhodopsin chemistry, Rhodopsin metabolism
- Abstract
Selective coupling of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-G
o protein. The conformation of the receptor is identical to all previous structures of active rhodopsin, including the complex with arrestin. Thus, rhodopsin seems to adopt predominantly one thermodynamically stable active conformation, effectively acting like a "structural switch," allowing for maximum efficiency in the visual system. Furthermore, our analysis of the well-defined GPCR-G protein interface suggests that the precise position of the carboxyl-terminal "hook-like" element of the G protein (its four last residues) relative to the TM7/helix 8 (H8) joint of the receptor is a significant determinant in selective G protein activation.- Published
- 2018
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49. GPCR-SAS: A web application for statistical analyses on G protein-coupled receptors sequences.
- Author
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Gómez Tamayo JC, Olivella M, Ríos S, Hoogstraat M, Gonzalez A, Mayol E, Deupi X, Campillo M, and Cordomí A
- Subjects
- Animals, Humans, Receptors, G-Protein-Coupled chemistry, Sequence Analysis, Protein methods, Software
- Abstract
G protein-coupled receptors (GPCRs) are one of the largest protein families in mammals. They mediate signal transduction across cell membranes and are important targets for the pharmaceutical industry. The G Protein-Coupled Receptors-Sequence Analysis and Statistics (GPCR-SAS) web application provides a set of tools to perform comparative analysis of sequence positions between receptors, based on a curated structural-informed multiple sequence alignment. The analysis tools include: (i) percentage of occurrence of an amino acid or motif and entropy at a position or range of positions, (ii) covariance of two positions, (iii) correlation between two amino acids in two positions (or two sequence motifs in two ranges of positions), and (iv) snake-plot representation for a specific receptor or for the consensus sequence of a group of selected receptors. The analysis of conservation of residues and motifs across transmembrane (TM) segments may guide the design of more selective ligands or help to rationalize activation mechanisms, among others. As an example, here we analyze the amino acids of the "transmission switch", that initiates receptor activation following ligand binding. The tool is freely accessible at http://lmc.uab.cat/gpcrsas/., Competing Interests: The authors have declared that no competing interests exist.
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- 2018
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50. Convergent evolution of tertiary structure in rhodopsin visual proteins from vertebrates and box jellyfish.
- Author
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Gerrard E, Mutt E, Nagata T, Koyanagi M, Flock T, Lesca E, Schertler GFX, Terakita A, Deupi X, and Lucas RJ
- Subjects
- Animals, HEK293 Cells, Humans, Molecular Dynamics Simulation, Phylogeny, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Cubozoa genetics, Evolution, Molecular, Rhodopsin chemistry, Rhodopsin genetics, Rhodopsin metabolism, Vision, Ocular genetics
- Abstract
Box jellyfish and vertebrates are separated by >500 million years of evolution yet have structurally analogous lens eyes that employ rhodopsin photopigments for vision. All opsins possess a negatively charged residue-the counterion-to maintain visible-light sensitivity and facilitate photoisomerization of their retinaldehyde chromophore. In vertebrate rhodopsins, the molecular evolution of the counterion position-from a highly conserved distal location in the second extracellular loop (E181) to a proximal location in the third transmembrane helix (E113)-is established as a key driver of higher fidelity photoreception. Here, we use computational biology and heterologous action spectroscopy to determine whether the appearance of the advanced visual apparatus in box jellyfish was also accompanied by changes in the opsin tertiary structure. We found that the counterion in an opsin from the lens eye of the box jellyfish Carybdea rastonii (JellyOp) has also moved to a unique proximal location within the transmembrane bundle-E94 in TM2. Furthermore, we reveal that this Schiff base/counterion system includes an additional positive charge-R186-that has coevolved with E94 to functionally separate E94 and E181 in the chromophore-binding pocket of JellyOp. By engineering this pocket-neutralizing R186 and E94, or swapping E94 with the vertebrate counterion E113-we can recreate versions of the invertebrate and vertebrate counterion systems, respectively, supporting a relatively similar overall architecture in this region of animal opsins. In summary, our data establish the third only counterion site in animal opsins and reveal convergent evolution of tertiary structure in opsins from distantly related species with advanced visual systems., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)
- Published
- 2018
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