25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

Conformational changes in the neurotensin peptide upon binding neurotensin receptor 1 may drive receptor activation

Not scheduled
4h
Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

Board: 555
Poster-Late Late-Breaking Posters

Speaker

Dr Fabian Bumbak (Indiana University)

Description

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 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). In all crystal structures, NT8-13 adopts an identical extended conformation with 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 is 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 structures. Using molecular dynamics (MD) simulations, we, and others, 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 indeed exists in two conformations. Two states were also observed for the free peptide but at different populations compared to the complex. Exchange rates between the two conformers are currently being measured. Taken together, our integrative approach has captured a peptide conformation not evident from crystal structures thus highlighting the importance of NT peptide dynamics for NTS1 activation.

Primary authors

Dr Fabian Bumbak (Indiana University) Mr Trayder Thomas (Monash University) Mr Billy Noonan-Williams (Monash University) Mrs Tasneem Vaid (The University of Melbourne) Mrs Fei Yan (The University of Melbourne) Mrs Alice Whitehead (The Florey Institute of Neuroscience and Mental Health) Mr Xuan Tan (The University of Alabama at Birmingham) Prof. Joshua Ziarek (Indiana University) Prof. Margaret Johnson (The University of Alabama at Birmingham) Prof. Ross Bathgate (University of Melbourne) Prof. David Chalmers (Monash University) Dr Daniel Scott (University of Melbourne) Prof. Paul Gooley (The University of Melbourne)

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