Encounter Complexes Between the N-terminal of Neurotensin with the Extracellular Loop 2 of the Neurotensin Receptor 1 Steer Neurotensin to the Orthosteric Binding Pocket.

Neurotensin (NT) is a linear disordered peptide that activates two different class A GPCRs, neurotensin receptor 1 (NTS) and NTS. Resolved structures of the complex of the C-terminal fragment of NT, NT8-13, with NTS shows the peptide takes a well-defined structure in the bound state. However, the mechanisms underlying NT recognition of NTS, and the conformational transition of NT upon binding NTS is an open question that if answered may aid discovery of highly selective drugs and reveal potential secondary binding sites on the surface of the receptor. Herein we investigated the interactions guiding NT to the orthosteric binding pocket of NTS by combining NMR experiments with kinetic analysis of the binding pathway using stopped-flow fluorescence and mutagenesis on both NT and NTS. We show the presence of transient structures in the middle part of NT that kinetically regulate the binding of NT to NTS. Moreover, our results indicate that the binding pathway of NT onto NTS is mediated via electrostatic interactions between the N-terminal region of NT with the extracellular loop 2 of NTS. These interactions induce backbone conformational changes in neurotensin similar to the bound-state neurotensin, suggesting that the N-terminal region of NT and these interactions should be considered for development of selective drugs against NTS.