μ-Opioid receptors (μOR) are G protein coupled receptors (GPCRs) that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive1 and agonist-induced active states (companion article) provide snapshots of the receptor at the beginning and end of a signaling event, but little is known about the dynamic sequence of events that span these two states. Here we report the use of solution-state NMR to examine the process of μOR activation. We obtained spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments (TM) 5 and 6, which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody revealing a weak allosteric coupling between the agonist binding pocket and the G protein coupling interface (TM5 and TM6) similar to what has been observed for the β2-adrenergic receptor2. Unexpectedly, in the presence of agonist alone, we observe larger spectral changes involving intracellular loop 1 (ICL1) and helix 8 (H8), when compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and ICL1 and/or H8 may play a role in G protein coupling specificity as has been suggested for other family A GPCRs.

Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.

Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the development of new isotopic labeling strategies has opened the possibility of exploiting NMR spectroscopy in the study of supra-molecular complexes with molecular weights of up to 1MDa. At the core of these isotopic labeling developments is the specific introduction of [(1)H,(13)C]-labeled methyl probes into perdeuterated proteins. Here, we describe the evolution of these approaches and discuss their impact on structural and biological studies. The relevant protocols are succinctly reviewed for single and combinatorial isotopic-labeling of methyl-containing residues, and examples of applications on challenging biological systems, including high molecular weight and membrane proteins, are presented.