Structure of a GPCR Ligand in Its Receptor-Bound State: Leukotriene B4 Adopts a Highly Constrained Conformation When Associated to Human BLT2

Abstract

G protein-coupled receptors (GPCRs) are key players in signal recognition and cell communication and are among the most important targets for drug development. Direct structural information on the conformation of GPCR ligands bound to their receptors is scarce. Using a leukotriene receptor, BLT2, expressed under a perdeuterated form in Escherichia coli , purified in milligram amounts, and folded to its native state using amphipols, we have solved, by (1)H NMR, the structure of receptor-bound leukotriene B4 (LTB4). Upon binding, LTB4 adopts a highly constrained seahorse conformation, at variance with the free state, where it explores a wide range of conformations. This structure provides an experimentally determined template of a pro-inflammatory compound for further pharmacological studies. The novel approach used for its determination could prove powerful to investigate ligand binding to GPCRs and membrane proteins in general.

Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor

“…The above 19 F NMR experiments may be performed 5-7°C higher in MNG-3 than in DDM over periods of days to weeks. It should be noted that there are other amphiphilic hosts such as amphipols (33)(34)(35) and nanodiscs (36,37) in which a similar if not better protein-stabilizing environment is attained, where exchange between functional states is possibly equally representative of physiological conditions. However, for the moment, new detergents such as MNG are a necessary evil for NMR purposes because the aggregate mass is minimal, and thus, T 2 is optimal for purposes of resolution and INEPT (insensitive nuclei enhanced by polarization transfer) transfers (31).…”

NMR structure and dynamics of the agonist dynorphin peptide bound to the human kappa opioid receptor

“…In aqueous solutions, peptides often exist as a dynamic ensemble of random coil conformers, with specific folds stabilized by organic solvents or micelles (35)(36)(37). Studies of liposome-bound peptides likewise indicate that nonpolar or membrane-like environments stabilize peptide structure (23,24,(38)(39)(40)(41). Schwyzer introduced the "membrane compartment concept" that postulated the membrane-bound state as part of the binding mechanism, thereby reducing the available peptide conformations toward an activating conformation in complex with receptor (21,22,25).…”

“…For many years, the direct analysis of the peptide-receptor complex was not possible due to the lack of suitable receptor preparations. Recently, several neurotensin-receptor complexes have been studied both by X-ray crystallography and solid-state NMR (11)(12)(13), and the structure of the Leukotriene B4 (a proinflammatory lipid mediator), in complex with the human BLT2 receptor, was determined by liquid-state NMR (41).…”

Exploring the Backbone of Enkephalins To Adjust Their Pharmacological Profile for the δ-Opioid Receptor

“…However, there is no evidence indicating that this crystal conformation corresponds to the active geometry of enkephalins. Indeed, induced fit requirements can dictate shapes that are quite distinct from crystal conformations (27). In light of this fact, it is possible that there is no β-turn in the active conformation of enkephalins or that a β-turn between amides 2 and 4 might have been selected by DOPR (28).…”