The mechanism of signal transduction mediated by G protein-coupled receptors is a subject of intense research in pharmacological and structural biology. Ligand association to the receptor constitutes a critical event in the activation process. Solution-state NMR can be amenable to high-resolution structure determination of agonist molecules in their receptor-bound state by detecting dipolar interactions in a transferred mode, even with equilibrium dissociation constants below the micromolar range. This is possible in the case of an inherent ultra-fast diffusive association of charged ligands onto a highly charged extracellular surface, and by slowing down the 1H–1H cross-relaxation by perdeuterating the receptor. Here, we demonstrate this for two fatty acid molecules in interaction with the leukotriene BLT2 receptor, for which both ligands display a submicromolar affinity.
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We are extremely grateful to Fabrice Giusti (UMR 7099) for the gift of deuterated surfactant amphipols. We thank Monica Zoppè (Scientific Visualization Unit, Inst. of Clinical Physiol., Pisa, Italy) for access to innovative visualization methods to illustrate the strong electrostatic potential gradient of BLT2. Particular thanks are due to Jean-Luc Popot (UMR 7099) and Jacky Marie (UMR 5247) for helpful comments on this manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS), Paris-7 University, and by grants from the E.U. (Specific Targeted Research Project LSHG-CT-2005-513770 IMPS Innovative tools for membrane protein structural proteomics) and from the French Ministry of Research (ANR-06-BLAN-0087 and ANR BLAN07-1_191475). L.J.C. is a 2009 recipient of Projets Exploratoires/ Premier Soutien (PEPS, Leukomotive project) from the CNRS.
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Catoire, L.J., Damian, M., Baaden, M. et al. Electrostatically-driven fast association and perdeuteration allow detection of transferred cross-relaxation for G protein-coupled receptor ligands with equilibrium dissociation constants in the high-to-low nanomolar range. J Biomol NMR 50, 191–195 (2011). https://doi.org/10.1007/s10858-011-9523-3
- Transferred NOE
- G protein-coupled receptor
- Signal transduction
- Structural biology