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

Abstract

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.

Structural and Functional Diversity among Agonist-Bound States of the GLP-1 Receptor

“…This conclusion is consistent with emerging evidence that conformational mobility in agonist and receptor can be functionally important in signal-transducing states of other GPCR-peptide complexes. 25,77,78 The mode of agonist mobility highlighted in this work may be evolutionarily conserved among peptide agonists of related Class B1 GPCRs; Gly at the fourth position from the N-terminus is found in glucagon, GLP-2 and several other hormones. 33 Other sites of essential mobility may be present in more distantly related hormones.…”

“…Dynorphin, a short opioid peptide, retains disorder when bound to the kappa opioid receptor, 77 and mobility of neurotensin residue Tyr-11 is required for activation of the cognate receptor. 78 Receptor activity-modifying proteins (RAMPs) also alter the conformational dynamics of the adrenomedullin receptors to effect changes in receptor phenotype. 74 Our findings are distinct from these precedents, however, in suggesting that at least two distinct states of an agonist-receptor complex may play important and complementary roles in the signal transduction mechanism.…”

Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function

“…A compound binding with a high affinity may freeze the inherent internal flexibility of NUPR1, impacting its activity in other ways. It is worth to note that a residual flexibility has been shown to be necessary to carry out the function of a number of proteins: for instance, neurotensin needs to maintain its flexibility around the crucial residue Tyr11 when the protein is bound to neurotensin receptor 1 [47], and the same applies for a whole nascent helix of dynein when it is bound to dynactin [48]. As a second possible explanation for the biological effects we observed, it has been recently argued that an effective strategy for drug discovery in IDPs relies in exploiting the entropic contributions due to the protein chain flexibility, which would increase the affinity towards ligands by shifting the population of disordered conformations [49,50].…”

The role of structural dynamics in GPCR‐mediated signaling

“…There is a growing body of evidence from NMR studies and 3DVA of cryo-EM data that peptide ligands can sample multiple conformations when bound to a receptor [183][184][185][186]. Even though the structural flexibility of peptides within the ligand-binding site has been shown to be important for receptor activation [184,186], it remains unclear how ligand dynamics affects the conformational landscape of receptors to modulate GPCR signaling. Answering this question will require the development of NMR or other spectroscopic methods that allow simultaneous analysis of ligand and receptor dynamics.…”