Oral Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Conformational flexibility of the bound neurotensin peptide may drive receptor activation (#46)

Fabian Bumbak 1 , Trayder Thomas 2 , Billy J Noonan-Williams 2 , Tasneem M Vaid 3 4 5 , Alice R Whitehead 5 , Xuan Tan 6 , Joshua J Ziarek 1 , Margaret A Johnson 6 , Ross AD Bathgate 3 5 , David K Chalmers 2 , Paul R Gooley 3 4 , Daniel J Scott 3 5
  1. Indiana University, Bloomington, INDIANA, United States
  2. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VICTORIA, Australia
  3. Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VICTORIA, Australia
  4. Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VICTORIA, Australia
  5. The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VICTORIA, Australia
  6. Department of Chemistry, The University of Alabama at Birmingham, Birmingham, Alabama, USA

Neurotensin (NT) is a 13 amino acid peptide expressed in the central nervous, gastro-intestinal and cardiovascular systems where it acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate, primarily through activation of the neurotensin receptor 1 (NTS1), a G Protein-Coupled Receptor (GPCR). Peptide ligands of GPCRs bind through complex, and possibly multiple modes for which there are few representative crystal structures. In the case of NTS1, eight inactive-state crystal structures of thermostabilized variants have been solved in complex with the high affinity 8-13 fragment of NT (NT8-13, RRPYIL)1-4 and two active-state Cryo-EM structures were recently solved in complex with a related agonist peptide (K-Ψ(CH2NH)-KPYIL)5. In all structures, the agonist peptide adopts a similar extended conformation of the C-terminal four residues (PYIL) buried deeply into the orthosteric site of the receptor. But the role of ligand conformational dynamics while bound to the orthosteric site remains unclear. We used competition saturation transfer difference (STD) NMR to investigate the binding pose of a low-affinity neurotensin peptide fragment (NT10-13) to a thermostabilized, signaling-competent NTS1 variant solubilized in detergent micelles. Epitope mapping of NT10-13 suggests that tyrosine 11 (Y11) binds the NTS1 orthosteric site in an alternate conformation than previously observed in crystal and Cryo-EM structures. Using molecular dynamics (MD) simulations, we, and others6, showed that Y11 can adopt two conformations on NTS1 binding. Furthermore, 13C HSQC spectra of a [U-13C,15N]-NT8-13:[U-2H,12C]-NTS1 complex revealed that the Y11 sidechain may indeed exist in multiple conformations when bound to the receptor. Next, we will measure exchange rates between the observed conformers and explore alternative NT8-13 labeling schemes to gather more information on the bound state. Taken together, our integrative approach has captured a peptide conformation not evident from crystal or Cryo-EM structures thus highlighting the importance of NT peptide dynamics for NTS1 activation.

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