Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1.

Cell-cell communication via endogenous peptides and their receptors is vital for controlling all aspects of human physiology and most peptides signal through G protein-coupled receptors (GPCRs). Disordered peptides bind GPCRs through complex modes for which there are few representative crystal structures. The disordered peptide neurotensin (NT) is a neuromodulator of classical neurotransmitters such as dopamine and glutamate, through activation of neurotensin receptor 1 (NTS). While several experimental structures show how NT binds NTS, details about the structural dynamics of NT during and after binding NTS, or the role of peptide dynamics on receptor activation, remain obscure. Here saturation transfer difference (STD) NMR revealed that the binding mode of NT fragment NT10-13 is heterogeneous. Epitope maps of NT10-13 at NTS suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS. Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS, residue Y11 can exist in two χ rotameric states, gauche plus (g) or gauche minus (g). Since only the g Y11 state is observed in all the structures solved to date, we asked if the g state is important for receptor activation. NT analogues with Y11 replaced with 7-OH-Tic were synthesized to restrain the dynamics of the side chain. P(OH-TIC)IL bound NTS with the same affinity as NT10-13 but did not activate NTS, instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS.