The serine hydrolase monoacylglycerol lipase (MGL) functions as the main metabolizing enzyme of 2-arachidonoyl glycerol, an endocannabinoid signaling lipid whose elevation through genetic or pharmacological MGL ablation exerts therapeutic effects in various preclinical disease models. To inform structure-based MGL inhibitor design, we report the direct NMR detection of a reversible equilibrium between active and inactive states of human MGL (hMGL) that is slow on the NMR time scale and can be modulated in a controlled manner by pH, temperature, and select point mutations. Kinetic measurements revealed that hMGL substrate turnover is rate-limited across this equilibrium. We identify a network of aromatic interactions and hydrogen bonds that regulates hMGL active-inactive state interconversion. The data highlight specific inter-residue interactions within hMGL modulating the enzymes function and implicate transitions between active (open) and inactive (closed) states of the hMGL lid domain in controlling substrate access to the enzymes active site.

We report the NMR solution structure of a synthetic 40-mer (T377-E416) that encompasses human cannabinoid receptor-1 (hCB1) transmembrane helix 7 (TMH7) and helix 8 (H8) [hCB1(TMH7/H8)] in 30% trifluoroethanol/H2O. Structural features include, from the peptide’s amino terminus, a hydrophobic α-helix (TMH7); a loop-like, eleven-residue segment featuring a pronounced Pro-kink within the conserved NPxxY motif; a short amphipathic α-helix (H8) orthogonal to TMH7 with cationic and hydrophobic amino-acid clusters; and an unstructured C-terminal end. The hCB1(TMH7/H8) NMR solution structure suggests multiple electrostatic amino-acid interactions, including an intrahelical H8 salt bridge and a hydrogen-bond network involving the peptide’s loop-like region. Potential cation-π and cation-phenolic OH interactions between Y397 in the TMH7 NPxxY motif and R405 in H8 are identified as candidate structural forces promoting interhelical microdomain formation. This microdomain may function as a flexible molecular hinge during ligand-induced hCB1-receptor conformer transitions.

Intramolecular hydrogen bonding is an important determinant of enzyme structure, catalysis, and inhibitor action. Monoacylglycerol lipase (MGL) modulates cannabinergic signaling as the main enzyme responsible for deactivating 2-arachidonoylglycerol (2-AG), a primary endocannabinoid lipid messenger. By enhancing tissue-protective 2-AG tone, targeted MGL inhibitors hold therapeutic promise for managing pain and treating inflammatory and neurodegenerative diseases. We report study of purified, solubilized human MGL (hMGL) to explore the details of hMGL catalysis by using two known covalent hMGL inhibitors, the carbamoyl tetrazole AM6701 and N-arachidonoylmaleimide (NAM), that act through distinct mechanisms. Using proton nuclear magnetic resonance spectroscopy (NMR) with purified wild-type and mutant hMGLs, we have directly observed a strong hydrogen-bond network involving Asp239 and His269 of the catalytic triad and neighboring Leu241 and Cys242 residues. hMGL inhibition by AM6701 alters this hydrogen-bonding pattern through subtle active-site structural rearrangements without influencing hydrogen-bond occupancies. Rapid carbamoylation of hMGL Ser122 by AM6701 and elimination of the leaving group is followed by a slow hydrolysis of the carbamate group, ultimately regenerating catalytically competent hMGL. In contrast, hMGL titration with NAM, which leads to cysteine alkylation, stoichiometrically decreases the population of the active-site hydrogen bonds. NAM prevents reformation of this network, and in this manner inhibits hMGL irreversibly. These data provide detailed molecular insight into the distinctive mechanisms of two covalent hMGL inhibitors and implicate a hydrogen-bond network as a structural feature of hMGL catalytic function.

An understanding of how conformational dynamics modulates function and catalysis of human monoacylglycerol lipase (hMGL), an important pharmaceutical target, can facilitate the development of novel ligands with potential therapeutic value. Here, we report the discovery and characterization of an allosteric, regulatory hMGL site comprised of residues Trp-289 and Leu-232 that reside over 18 Å away from the catalytic triad. These residues were identified as critical mediators of long-range communication and as important contributors to the integrity of the hMGL structure. Nonconservative replacements of Trp-289 or Leu-232 triggered concerted motions of structurally distinct regions with a significant conformational shift toward inactive states and dramatic loss in catalytic efficiency of the enzyme. Using a multimethod approach, we show that the dynamically relevant Trp-289 and Leu-232 residues serve as communication hubs within an allosteric protein network that controls signal propagation to the active site, and thus, regulates active-inactive interconversion of hMGL. Our findings provide new insights into the mechanism of allosteric regulation of lipase activity, in general, and may provide alternative drug design possibilities.