SummaryNeurotensin (NT) is a 13 amino acid peptide that functions as both a neurotransmitter and a hormone through activation of the neurotensin receptor NTS1, a G protein-coupled receptor (GPCR). In the brain, NT modulates activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake, and in the gut NT regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of NTS1 in an active-like state, bound to NT8-13, the C terminal portion of NT responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTS1 in an extended conformation nearly perpendicular to the membrane plane with the C-terminus oriented towards the receptor core. Our findings provide the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.

G protein-coupled receptors (GPCRs) mediate the perception of smell, light, taste, and pain. They are involved in signal recognition and cell communication and are some of the most important targets for drug development. Because currently no direct structural information on high-affinity ligands bound to GPCRs is available, rational drug design is limited to computational prediction combined with mutagenesis experiments. Here, we present the conformation of a high-affinity peptide agonist (neurotensin, NT) bound to its GPCR NTS-1, determined by direct structural methods. Functional receptors were expressed in Escherichia coli, purified in milligram amounts by using optimized procedures, and subsequently reconstituted into lipid vesicles. Solid-state NMR experiments were tailored to allow for the unequivocal detection of microgram quantities of 13 C, 15 N-labeled NT(8 -13) in complex with functional NTS-1. The NMR data are consistent with a disordered state of the ligand in the absence of receptor. Upon receptor binding, the peptide undergoes a linear rearrangement, adopting a ␤-strand conformation. Our results provide a viable structural template for further pharmacological investigations.G protein-coupled receptors (GPCRs) are integral membrane proteins involved in a number of important physiological processes, including sensory transduction, mediation of hormonal activity, and cell-to-cell communication (for reviews, see refs. 1 and 2). More than 1,000 different GPCRs have been identified, and many of them have been implicated as major therapeutic routes to the treatment of human diseases (3). Despite the striking clinical relevance of GPCRs, only one high-resolution structure (rhodopsin) is available (4, 5). The diversity among endogenous GPCR ligands is exceptional. Small molecule ligands such as biogenic amines have been proposed to bind within the hydrophobic core of their respective receptors. In contrast, mutational mapping of ligand-binding sites in peptide receptors indicates that extracellular domains are also involved in ligand recognition (2). To date, direct structural information regarding ligand binding and GPCR activation is very limited (6). Targeted drug design is restricted to computational methods and other ligand design approaches to find and optimize lead compounds (7). The precise knowledge of receptor-bound ligand structures would substantially aid the development of tailor-made medicines.Neurotensin (NT) is a 13-aa peptide (8) that is involved in a variety of neuromodulatory functions in the central and peripheral nervous system (9). NT binds to its GPCRs, NT type I receptor (NTS-1) and NTS-2 (10 -13). The levocabastineinsensitive NTS-1 (10-12) interacts with the agonist NT with high (i.e., subnanomolar) affinity. Similar observations were made for the N-terminally truncated form of rat NTS-1 when expressed as a maltose-binding protein (MBP) fusion in Escherichia coli (14, 15) and purified in the presence of detergents (14, 16). Notably, not only the full-length peptide, but also the C-...