Oral Presentation 23rd International Society of Magnetic Resonance Conference 2023

The neurotensin peptide agonist retains conformational flexibility upon binding to neurotensin receptor 1 (#55)

Fabian Bumbak 1 2 , Wijnand JC van der Velden 3 , Skylar Zemmer 1 , Hongwei Wu 4 , Piotr A Mroz 4 , Mingzhe Pan 1 , Scott A Robson 1 , Brennan A Murphy 4 , Richard D DiMarchi 4 , Yohey Miyanoiri 5 6 , Masatsune Kainosho 5 7 , Nagarajan Vaidehi 3 , Joshua J Ziarek 1
  1. Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA
  2. Present address: ARC Centre for Cryo-electron Microscopy of Membrane Proteins and Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VICTORIA, Australia
  3. Beckman Research Institute, Computational and Quantitative Medicine, City of Hope, Duarte, California, USA
  4. Department of Chemistry, Indiana University, Bloomington, Indiana, USA
  5. Structural Biology Research Center, Graduate School of Science, Nagoya University, Chikusa, Nagoya, Japan
  6. Present address: Institute for Protein Research, Osaka University, Suita, Osaka, Japan
  7. Present address: Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo, Japan

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, numerous inactive-state and active-state Cryo-EM structures were determined in complex with either the high affinity 8-13 fragment of NT (NT8-13, RRPYIL) or a related agonist peptide (K-Ψ(CH2NH)-KPYIL)1-10. In all structures, the agonist peptide adopts an identical extended backbone conformation with the C-terminal four residues (PYIL) buried deeply into the orthosteric binding site of the receptor. But the role of ligand conformational dynamics while bound to the orthosteric site is unclear. A previous STD-NMR study of the low-affinity fragment NT10-1311 and molecular dynamics (MD) simulations of NT8-1312 bound to NTS1 suggested that particularly tyrosine 11 (Y11) of the ligand may adopt multiple conformations in the bound state. [U-13C, 15N]-labeled neurotensin fragment NT8-13 (RRPYIL) in combination with [U-2H]-labeled, thermostabilized NTS1 enabled us to capture receptor-bound conformational heterogeneity of the peptide by solution NMR. 13C CT-HSQC spectra suggest two-state ensembles for the methyl resonances of ligand residues I12 and L13 deeply buried in the binding pocket, with one set resembling the receptor-free peptide. Y11δ and Y11ε resonances reveal three and two distinct chemical environments, respectively, indicating that either ring-flipping or Chi1 angle interconversion occur at a slow (millisecond to second timescale) rate when the peptide is bound to the receptor. We further show that P10 cis/trans isomerization observed for free NT8-13 is likely absent in the receptor bound state. Taken together, our data shows that NTS1-bound NT8-13 is characterized by at least two slowly interconverting conformations.

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