Project

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Structural characterisation of ligand-induced conformational changes in G protein coupled receptors

English title Structural characterisation of ligand-induced conformational changes in G protein coupled receptors
Applicant Veprintsev Dmitry
Number 135754
Funding scheme Project funding
Research institution GPCR Signalling Biomolecular Research Paul Scherrer Institute
Institution of higher education Paul Scherrer Institute - PSI
Main discipline Molecular Biology
Start/End 01.05.2011 - 31.10.2014
Approved amount 331'000.00
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All Disciplines (2)

Discipline
Molecular Biology
Biophysics

Keywords (5)

G protein coupled receptors; stability; structure; g protein; ligand binding

Lay Summary (English)

Lead
Lay summary

laysummary

G protein-coupled receptors (GPCRs) are trans-membrane proteins located at the cell surface, which sense extracellular neural, endocrine, olfactory and visual signals and convert them into appropriate intra-cellular responses. When activated by an agonist, GPCRs in turn activate G proteins, which initiate signalling cascades inside the cell. Over a half of current drugs target these proteins, highlightening the pharmacological relevance of the GPCR protein family. Some GPCRs can recognise several ligands, with diverse physiological and pharmacological outputs. One of the most important questions in structural biology of GPCRs is to understand how ligands can induce distinct conformations and activate different downstream signalling pathways.

The aim of the proposed research is to identify the number of distinct conformational states, to structurally characterise ligand-induced conformational changes in GPCRs and to engineer conformationally stabilised variants for subsequent biophysical and structural studies. The project will focus on cannabinoid CB2 and ß1-adrenoreceptors, which are involved in regulation of peripheral immune system and regulation of blood flow via muscle contraction, respectively. Specifically, the project aims to identify conformational changes induced by agonist ligands using a novel protein engineering approach that combines alanine scanning with conformational f-value analysis, complemented by ligand binding and activity measurements. Comparison of the conformational changes caused by a spectrum of ligands with different activities, from antagonists to agonists, will provide a mechanistic view of GPCR signal transduction and facilitate rational drug design.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Universal allosteric mechanism for Gα activation by GPCRs.
Flock Tilman, Ravarani Charles N J, Sun Dawei, Venkatakrishnan A J, Kayikci Melis, Tate Christopher G, Veprintsev Dmitry B, Babu M Madan (2015), Universal allosteric mechanism for Gα activation by GPCRs., in Nature, 524(7564), 173-9.
Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein.
Maeda Shoji, Sun Dawei, Singhal Ankita, Foggetta Marcello, Schmid Georg, Standfuss Joerg, Hennig Michael, Dawson Roger J P, Veprintsev Dmitry B, Schertler Gebhard F X (2014), Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein., in PloS one, 9(6), 98714-98714.
AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis.
Sun Dawei, Ostermaier Martin K, Heydenreich Franziska M, Mayer Daniel, Jaussi Rolf, Standfuss Joerg, Veprintsev Dmitry B (2013), AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis., in PloS one, 8(10), 78878-78878.
G protein-coupled receptor activation based on X-ray structural studies.
Veprintsev Dmitry B., Deupi Xavier, Standfuss Joerg, Schertler Gebhard F.X. (2013), G protein-coupled receptor activation based on X-ray structural studies., in Roberts Gordon (ed.), Springer, Germany, 877-881.
Insights into congenital stationary night blindness based on the structure of G90D rhodopsin.
Singhal Ankita, Ostermaier Martin K, Vishnivetskiy Sergey A, Panneels Valérie, Homan Kristoff T, Tesmer John J G, Veprintsev Dmitry, Deupi Xavier, Gurevich Vsevolod V, Schertler Gebhard F X, Standfuss Joerg (2013), Insights into congenital stationary night blindness based on the structure of G90D rhodopsin., in EMBO reports, 14(6), 520-6.
Towards structural studies of ligand-induced conformational changes in arginine-vasopressin V2 receptor
Milic D., Heydenreich F., Veprintsev D. B. (2013), Towards structural studies of ligand-induced conformational changes in arginine-vasopressin V2 receptor, 280, 280.
Probing Gαi1 protein activation at single-amino acid resolution
Sun Dawei, Flock Tilman, Deupi Xavier, Maeda Shoji, Matkovic Milos, Mendieta Sandro, Mayer Daniel, Dawson Roger J., Schertler Gebhard FX, Babu M. Madan, Veprintsev Dmitry B., Probing Gαi1 protein activation at single-amino acid resolution, in Nat Struct Mol Biol.

Collaboration

Group / person Country
Types of collaboration
Madan Babu group/Laboratory of Molecular Biology Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Reinhard Grisshammer group United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
3rd Annual Meeting of the GDR 3545 “From Physiology to Drugs: RCPG-PHYSIO-MED Poster Towards structural studies of cannabinoid CB2 and vasopressin V2 receptors 20.10.2014 Montpellier, France Heydenreich Franziska; Veprintsev Dmitry;
GLISTEN Talk given at a conference Probing Gαi1 protein activation at single amino acid resolution 01.10.2014 Budapest, Hungary Veprintsev Dmitry;
Keystone conference "G protein coupled receptors" Poster Phi-value analysis of a G protein 04.04.2014 Utah, United States of America Veprintsev Dmitry;
GPCR Workshop Poster Structural Characterization of GPCR-mediated Conformational Changes in the Alpha Subunit of a G Protein 01.12.2013 HAwaii, United States of America Sun Dawei;


Associated projects

Number Title Start Funding scheme
132815 Linking G protein-coupled receptor structure to signaling output 01.12.2010 Project funding
141898 NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins 01.12.2012 Sinergia
159748 Structural and biophysical pharmacology of cannabinoid CB2 and vasopressin V2R G protein-coupled receptors 01.09.2015 Project funding
146520 Structural basis of G protein-coupled receptor activation by biased ligands 01.04.2013 Project funding

Abstract

G protein-coupled receptors (GPCRs) are trans-membrane proteins that sense extracellular neural, endocrine, olfactory and visual signals and convert them into appropriate intra-cellular responses. When activated by an agonist, GPCRs in turn activate G proteins, which initiate signalling cascades inside the cell. Some GPCRs can recognise several ligands, with diverse physiological and pharmacological outputs. Over a half of current drugs target these proteins, highlightening the pharmacological relevance of the GPCR protein family. One of the most important questions in structural biology of GPCRs is to understand how ligands can induce distinct conformations and activate different downstream signalling pathways. An understanding of conformational changes associated with GPCR activation will provide a mechanistic view of GPCR activation and facilitate rational drug design. The aim of the proposed research is to identify the number of distinct conformational states, to structurally characterise ligand-induced conformational changes in GPCRs and to engineer conformationally stabilised variants for subsequent biophysical and structural studies. The project will focus on cannabinoid receptor CB2 and ?1-adrenoreceptor. Specifically, I propose to: a) Identify conformational changes induced by agonist ligands using a novel protein engineering approach that combines alanine scanning with conformational ?-value analysis, complemented by ligand binding and activity measurements. b) Analyze the data to obtain a dynamic view of the conformational changes in GPCR and model the structure of ligand-bound active states. c) Engineer by rational mutagenesis a series of proteins stabilised in specific conformations, which will facilitate structure determination and biophysical characterisation of the receptor and of its interaction with G proteins. d) Comparison of the conformational changes caused by a spectrum of ligands with different activities, from antagonists to agonists, will give us a comprehensive mechanistic insight into the activation mechanism. Due to the high sequence conservation, the result of this work will have wide implications for the GPCR family of proteins. In addition, this general approach could also be extended to other “difficult” membrane proteins.
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