Neurotensin receptor 1 (NTS1), a class A G protein-coupled receptor, plays critical roles in functions of the central nervous system and gastrointestinal tract. NTS1 is activated by the endogenous 13-residue peptide neurotensin, NT (pELYENKPRRPYIL). Although NT shows a specific conformation in the NTS1 bound state in crystal and NMR structures, molecular dynamic simulations in our lab and by others, supported by STD analyses, revealed the peptide remains flexible in the bound state. Notably, Y11 exists in both the +/- gauche states for the χ1 rotamer and restriction of this sidechain reduces the efficacy of NT.
To investigate this further, we generated NT analogues containing fluorinated mimetics of tyrosine, trifluoromethylphenylalanine tfmF, at position 11 (Y11tfmF-NT) and employed sensitive 19F NMR to probe the correlation of Y11 conformation with receptor activation. Y11tfmF-NT was able to bind and activate NTS1, albeit with weaker affinity and potency to NT, indicating that Y11tfmF-NT could be used to understand NTS1 activation with 19F NMR. 19F NMR spectra of Y11tfmF-NT was acquired in the free state, or in complex with purified rat NTS1 and revealed that Y11 populated two conformations, a major population of ~85% and a minor population of ~15%. The high chemical shift dispersion and receptivity of 19F nuclei gives us the opportunity to measure the exchange rate between these two states, which is not feasible by conventional isotope labelling methods. Saturation transfer experiments indicated two conformations to be in very slow exchange (1 <s-1). Furthermore, mutation of key residues in the peptide and the receptor altered these rates, supporting the hypothesis that the conformational dynamics of Y11 is critical to enNTS1 activation.
The data presented here is providing insight into the mechanism of activation of NTS1 by peptide ligands that will consequently pave the way for rational drug design against NTS1.