Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

Unravelling the mechanism of neurotensin recognition by neurotensin receptor 1 (#201)

Kazem KA Asadollahi 1 , Sunnia SR Rajput 2 , Andrew AZ Zhang 3 , Ching-Seng Ang 2 , Guy Jameson 4 , Thomas Weikl 5 , Daniel DJS Scott 3 , Paul PRG Gooley 1 2
  1. Department of Biochemistry and Pharmacology, University of Melbourne, PARKVILLE, VIC, Australia
  2. Bio21 Institute, University of Melbourne, PARKVILLE, VIC, Australia
  3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, PARKVILLE, VIC, Australia
  4. School of Chemistry, University of Melbourne, Melbourne, Victoria, Australia
  5. Department of Molecular Biosystems, Max Planck Institute of Colloids and Interfaces, Postdam, Brandenburg, Germany

G-protein coupled receptors (GPCRs) are the largest superfamily of membrane receptors and the largest group of targetable membrane proteins. Indeed, 30-40% of marketed drugs target GPCRs [1]. However, the mechanisms governing GPCR activation, which is crucial for the development of tailored drugs, are still unknown. The proposed mechanism underlying GPCR activation, derived largely from analysis of GPRC structure and dynamics, is conformational selection, in which the apo receptor exists in a conformational pre-equilibrium between different active and inactive states [2]. Ligand binding to the GPCR selects for a particular conformation, in an efficacy dependent manner that shifts this equilibrium accordingly. However, conformational selection is insufficient to explain the complex pharmacological behaviour of GPCRs. We focused on the mechanism of ligand recognition and activation of neurotensin receptor 1 (NTS1), a class A GPCR that plays critical roles in the central nervous system and gastrointestinal tract. NTS1 is activated by the endogenous linear 13-residue peptide neurotensin, NT (pELYENKPRRPYIL). Despite several structures of NTS1 being available, the mechanism underlying NTS1 activation is still unknown. Our biophysical and kinetic studies on the mechanism of neurotensin recognition by neurotensin receptor 1 (NTS1) using 19F-NMR, hydrogen-deuterium exchange mass spectrometry and stopped-flow fluorescence spectroscopy revealed slow-exchanging conformational heterogeneity on the extracellular surface of ligand-bound NTS1 that follows an induced fit mechanism, in which conformational changes occur after neurotensin binding via formation of encounter complexes between NT and NTS1. These intermediate states explain the kinetic bias model [3] and sequential activation model [4] in GPCR activation and can open avenues for development of allosteric or bitopic ligands against NTS1.This approach is applicable to other GPCRs to provide insight into the kinetic regulation of ligand recognition by GPCRs.

  1. 1. Garland SL. Are GPCRs still a source of new targets? J Biomol Screen 18, 947-966 (2013). 2. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection 3. Klein Herenbrink C, et al. The role of kinetic context in apparent biased agonism at GPCRs. Nat Commun 7, 10842 (2016). 4. Grundmann M, et al. A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor. Cell Chem Biol 23, 392-403 (2016).