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 (NTS₁). While several experimental structures show how NT binds NTS₁, details about the structural dynamics of NT during and after binding NTS₁, 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 NTS₁ suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS₁. Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS₁, residue Y11 can exist in two χ₁ 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 NTS₁ with the same affinity as NT10-13 but did not activate NTS₁, instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS₁.