Probing the environment of neurotensin whilst bound to the neurotensin receptor by solid state NMR

Abstract

A functionally active analogue of neurotensin, neurotensin(8^13), has been observed whilst bound to the agonistbinding site of the rat neurotensin receptor by nuclear magnetic resonance (NMR). Through the application of slow magic angle sample spinning and high-power proton decoupling, sufficient resolution and sensitivity were obtained in the carbon-13 spectrum to allow an assignment of many of the side chain resonances arising from uniformly carbon-13/nitrogen-15-labelled neurotensin(8^13) whilst bound to the neurotensin receptor. Significant perturbations in carbon-13 chemical shift were observed upon the binding of the neurotensin(8^13) to the receptor. Most importantly significant shifts were observed in both the carboxy terminus and tyrosine side chain of the neurotensin(8^13), suggesting that these sites are important in the interaction of the neurotensin with the agonist-binding site on the neurotensin receptor. Conversely, no perturbations were observed for the carbon-13 sites within the guanidinium groups of the arginine side chains, indicating little interaction with the receptor-binding site, or a shielding of the local environment by the surrounding nitrogen atoms. These NMR observations lend further support to previous structure^activity studies, site-directed mutagenesis and modelling studies of the agonist-binding site of the neurotensin receptor, from which the same specific residues for which NMR perturbations were observed are important for neurotensin receptor activation by neurotensin. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Neurotensin receptor type 1: Escherichia coli expression, purification, characterization and biophysical study

“…Rat NTS1 has been expressed previously as a protein fusion with MBP (maltose-binding protein) and TrxA (thioredoxin)-His 10 in the Escherichia coli DH5α strain [6][7][8]. Expression trials of the NTS1 construct were carried out in E. coli DH5α and BL21(DE3) cells, as well as C41(DE3) and C43(DE3)pREP4, two mutant strains derived from BL21(DE3) which have proved useful for the overexpression of several membrane and toxic proteins [9].…”

The conformation of neurotensin bound to its G protein-coupled receptor

“…6). A recent NMR study in the presence of detergent reported NT(8-13) chemical shift changes upon receptor interaction (18,19). However, the mode of NT binding to its GPCR NTS-1 remains unknown.…”

Intermolecular interactions between the neurotensin and the third extracellular loop of human neurotensin 1 receptor

“…Despite the physiological importance of NT, very few NMR studies so far have examined the structure of NT when free in solution (Coutant, Curmi, Toma, & Monti, 2007;Nieto, Rico, Santoro, Herranz, & Bermero, 1986;Richard, Vitrac, Merillon, & Monti, 2005;Xu & Deber, 1991) or in interaction with NTSs, and only two have used NMR-MAS (Luca et al, 2003;Williamson, Bains, Chung, Cooke, & Watts, 2002). However, such knowledge would provide insights into the specificity of NT: NTSs binding and would help in developing new molecules or selecting those with putative pharmacological interest among compound libraries.…”

“…While two NMR-MAS studies investigated the structure of the NT:NTS1 interaction, the authors only used the NT(8-13) fragment. In the first work using 13 C chemical shifts of NT(8-13), the role of the Tyr 11 side chain was underlined for NT interaction with the receptor, but the authors did not propose any threedimensional (3D) structure (Williamson et al, 2002). In the second, the authors used an indirect method based on TALOS and the 13 C chemical shift index (CSI) for NT(8-13) to suggest a β-strand conformation for the NT(8-13) bound fragment (Luca et al, 2003).…”