Project

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NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins

Applicant Schertler Gebhard
Number 141898
Funding scheme Sinergia
Research institution GPCR Signalling Biomolecular Research Paul Scherrer Institute
Institution of higher education Paul Scherrer Institute - PSI
Main discipline Biophysics
Start/End 01.12.2012 - 30.11.2015
Approved amount 1'214'803.00
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All Disciplines (4)

Discipline
Biophysics
Biochemistry
Structural Research
Molecular Biology

Keywords (7)

nuclear magnetic resonance; NMR; GPCR; signalling; structure; dynamics; structural biology

Lay Summary (English)

Lead
Lay summary

G protein-coupled receptors (GPCRs) are membrane proteins that sense extracellular neural and endocrine signals and activate a number of intracellular signalling pathways. Over 30% of the drugs used in medicine today modulate the activity of GPCRs. Recently, GPCR drugs have been developed that instead of fully activating or deactivating the receptor, bias the receptor towards activation of distinct intracellular pathways. These biased drugs offer improved therapeutic potential by reducing the side-effects from unwanted modulation of off-target signalling pathways.

Knowing the dynamics of biased drug-receptor complexes will lead to a deeper understanding of the molecular basis of this biased signalling. In this SINERGIA project, we are extending our successful electron and x-ray crystallographic approaches to structure determination of GPCRs to probe their dynamic behaviour by NMR. This is a challenging task that requires a combination of our expertise at the Laboratory for Biomolecular Research at the Paul Scherrer Institut in expression, stabilisation, handling and structural biology of GPCRs with the expertise in NMR at the Department of Biology at ETH Zürich and the Biozentrum at the University of Basel.

The important questions we are going to address are the number of conformational states of the receptor, the dynamics of their exchange, and the effect of ligands and signalling proteins on the conformational equilibrium. In particular, we propose: a) to label GPCRs for NMR analysis; b) to obtain a dynamic view of the conformational changes in GPCRs induced by a range of ligands with different activities including agonists, antagonists and biased ligands; c) to investigate the interaction of receptors with their downstream signalling partners (G proteins and arrestins); d) to identify the molecular basis for signalling selectivity via a comparative NMR and biophysical analysis of receptor binding to the various ligands, G proteins and arrestin.

We anticipate that NMR will become an extremely valuable technique to study structural dynamics of GPCRs. Our results will help future efforts in drug design and are expected to have wide implications for signal transduction by GPCRs in general.

 
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor.
Isogai Shin, Deupi Xavier, Opitz Christian, Heydenreich Franziska M, Tsai Ching-Ju, Brueckner Florian, Schertler Gebhard F X, Veprintsev Dmitry B, Grzesiek Stephan (2016), Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor., in Nature, 530(7589), 237-41.
Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis.
Chakrabarti Kalyan S, Agafonov Roman V, Pontiggia Francesco, Otten Renee, Higgins Matthew K, Schertler Gebhard F X, Oprian Daniel D, Kern Dorothee (2016), Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis., in Cell reports, 14(1), 32-42.
An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts.
Opitz Christian, Isogai Shin, Grzesiek Stephan (2015), An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts., in Journal of biomolecular NMR, 62(3), 373-85.
Large-scale production and protein engineering of G protein-coupled receptors for structural studies.
Milić Dalibor, Veprintsev Dmitry B (2015), Large-scale production and protein engineering of G protein-coupled receptors for structural studies., in Frontiers in pharmacology, 6, 66-66.
Stabilization of G protein-coupled receptors by point mutations.
Heydenreich Franziska M, Vuckovic Ziva, Matkovic Milos, Veprintsev Dmitry B (2015), Stabilization of G protein-coupled receptors by point mutations., in Frontiers in pharmacology, 6, 82-82.

Collaboration

Group / person Country
Types of collaboration
Prof Assaf Friedler Israel (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
XXI Swiss NMR Symposium Talk given at a conference Using backbone NMR to follow signaling in a eukaryotic GPCR + Plans for a 1.2 GHz solution NMR instrument in Switzerland 04.02.2016 Lausanne, Switzerland Grzesiek Stephan;
International workshop on RRR, Kyoto University Talk given at a conference Quantitative analysis of dynamics, structure and function of folded and unfolded proteins by advanced solution NMR techniques 12.12.2015 Kyoto, Japan Grzesiek Stephan;
GPCR Workshop Talk given at a conference Solution NMR analysis of the β1-adrenergic receptor 01.12.2015 Kona, Hawaii, United States of America Veprintsev Dmitry; Isogai Shin;
Kyoto University JGP Seminar Talk given at a conference Insights into protein structure, dynamics and function from NMR using Abelson kinase and the b1-adrenergic G protein coupled receptor as examples 26.11.2015 Kyoto, Japan Grzesiek Stephan;
GLISTEN European GPCR network conference Talk given at a conference Conformational dynamics in GPCR signalling 02.10.2015 Budapest, Hungary Veprintsev Dmitry; Milic Dalibor;
29th RegioMeeting on Structural Biology Talk given at a conference Backbone NMR reveals allosteric signaling networks in the β1-adrenergic receptor 23.09.2015 Engelberg, Switzerland Isogai Shin;
Euromar 2014 Talk given at a conference Biomolecular dynamics, interactions and function studied by quantitative NMR methods 29.06.2015 Turich, Switzerland Isogai Shin; Grzesiek Stephan;
GLISTEN, Talk given at a conference Protein backbone NMR of the β1-adrenergic receptor 01.04.2015 Allschwil, Switzerland Grzesiek Stephan; Isogai Shin; Schertler Gebhard; Veprintsev Dmitry; Milic Dalibor; Heydenreich Franziska;
Euromar 2014 Talk given at a conference Solution NMR analysis of beta1-adrenergic receptor 29.06.2014 Zurich, Switzerland Grzesiek Stephan; Isogai Shin; Veprintsev Dmitry;


Communication with the public

Communication Title Media Place Year
Media relations: print media, online media Probing what sets the heart racing PSI Press release International Western Switzerland Italian-speaking Switzerland German-speaking Switzerland 2016

Associated projects

Number Title Start Funding scheme
165219 Functional characterisation of Vasopressin V2 receptor at single amino acid resolution 01.01.2016 Doc.Mobility
150814 Purchase of a 900 MHz high-resolution NMR instrument 01.12.2013 R'EQUIP
173089 Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology 01.04.2017 Project funding (Div. I-III)
173335 Analyzing protein dynamics in receptor signaling relevant to pharmacology 01.06.2017 Project funding (Div. I-III)
135754 Structural characterisation of ligand-induced conformational changes in G protein coupled receptors 01.05.2011 Project funding (Div. I-III)
132857 Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology 01.10.2010 Project funding (Div. I-III)
189379 Structure determination of protein-RNA complexes involved in pre-mRNA splicing and translation regulation 01.10.2019 Project funding (Div. I-III)
149927 Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology 01.10.2013 Project funding (Div. I-III)
168031 Elucidating allosteric signal transmission in the beta1-adrenergic receptor 01.04.2017 Ambizione
153145 Serial femtosecond crystallography of GPCR signaling systems 01.06.2014 Project funding (Div. I-III)
132815 Linking G protein-coupled receptor structure to signaling output 01.12.2010 Project funding (Div. I-III)
149921 NMR structure determination of protein-RNA complexes involved in pre-mRNA splicing and translation regulation 01.10.2013 Project funding (Div. I-III)

Abstract

G protein-coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular neural, endocrine, olfactory and visual signals across the plasma membrane. When activated by light or a ligand GPCRs undergo a conformational change leading to activation of a G protein, arrestin and other signaling pathways. Over 30% of compounds used in medicine today modulate the activity of GPCRs. Some GPCRs can bind a range of ligands, some of which, instead of fully activating the receptor, rather bias the signal transduction cascade toward distinct intracellular pathways. The currently available X-ray structures of several GPCRs and of a ?2-adrenoreceptor/Gs signaling complex provide a frozen snapshot of a signal transduction event, but do not fully explain how the ligand selectivity is achieved, and how the ligand binding results in preferential binding of a specific signaling protein such as Gi, Gs or arrestin. Understanding the precise structural and dynamic nature of these phenomena is critical to knowing the mechanism of GPCR activation and will help further development of pharmaceuticals with desired pharmacological properties. We propose here to provide the missing dynamical information on GPCR function by Nuclear magnetic resonance spectroscopy (NMR), which shall determine the conformational changes and dynamics of the receptors and signaling complexes in solution. This should then a yield a comprehensive view of GPCR activation of and signal transduction. We will initially focus on rhodopsin and ?1-adrenoreceptor (?1AR), as well as their signaling complexes with G proteins and arrestin, and later apply developed expertise to the vasopressin receptor V2R and its signaling complexes.In particular, we propose: a) to label GPCRs for NMR analysis; b) to obtain a dynamic view of the conformational changes in GPCRs induced by a range of ligands with different activities including agonists, antagonists and biased ligands; c) to investigate the effect of G proteins and arrestin binding on the receptors; d) to identify the molecular basis for signaling selectivity via a comparative NMR and biophysical analysis of receptor binding to the various ligands, G proteins and arrestin.Due to the high sequence conservation among G protein-coupled receptor proteins our results are expected to have wide implications for signal transduction by GPCRs in general.
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