Substrate Report for: AHMMCE

Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger 2-arachidonoylglycerol. Agents that carbamylate hMGLs catalytic Ser(122) constitute a leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGL's conformational responses to two potent carbamylating inhibitors, AM6580 (irreversible) and AM6701 (slowly reversible). A dynamic, solvent-exposed lid domain is characteristic of hMGL's solution conformation. Both hMGL inhibitors restricted backbone enzyme motility in the active-site region and increased substrate binding-pocket solvent exposure. Covalent reaction of AM6580 with hMGL generates a bulkier carbamylated Ser(122) residue as compared to the more discrete Ser(122) modification by AM6701, a difference reflected in AM6580's more pronounced effect upon hMGL conformation. We demonstrate that structurally distinct carbamylating hMGL inhibitors generate particular conformational ensembles characterized by region-specific hMGL dynamics. By demonstrating the distinctive influences of two hMGL inhibitors on enzyme conformation, this study furthers our understanding at the molecular level of the dynamic features of hMGL interaction with small-molecule ligands.

The membrane-associated serine hydrolase, monoacylglycerol lipase (MGL), is a well-recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGL's membrane association upon its structure-function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge-neutral phospholipid bilayer nanodiscs enhanced hMGL's kinetic properties [apparent maximum velocity (Vmax ) and substrate affinity (Km )]. Hydrogen exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL-nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid-domain helix alpha4 and neighboring helix alpha6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzyme's substrate-binding channel. Covalent modification of membrane-associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane-associated topology of active and inhibited, covalently-modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzyme's conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity.

In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu(169);Leu(176))-to-Ser(Ser(169);Ser(176)) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser(122) with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser(122) residue as compared to that of Cys(122) in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys(122) residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser(122), and the mechanisms underlying hMGL inhibition by different classes of small molecules.

Human alpha/beta hydrolase domain 6 (hABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol (2-AG), a potent agonist at both cannabinoid CB1 and CB2 receptors. In vivo modulation of ABHD6 expression has been shown to have potential therapeutic applications, making the enzyme a promising drug target. However, the lack of structural information on hABHD6 limits the discovery and design of selective inhibitors. We have performed E. coli expression, purification and activity profiling screening of different hABHD6 constructs and identified a truncated variant without N-terminal transmembrane (TM) domain, hDelta29-3-ABHD6, as the most promising protein for further characterization. The elimination of the TM domain did not affect 2-AG or fluorogenic arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE) substrates hydrolysis, suggesting that the TM is not essential for enzyme catalytic activity. The hDelta29-3-ABHD6 variant was purified in a single step using Immobilized Metal Affinity Chromatography (IMAC), in-solution trypsin digested, and proteomically characterized by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The N-terminal peptide without methionine was identified indicating on a post-translational modification of the recombinant protein. The mechanism of inhibition of hABHD6 with AM6701 and WWL70 covalent probes was elucidated based on MS analysis of trypsin digested hABHD6 following the Ligand Assisted Protein Structure (LAPS) approach. We identified the carbamylated peptides containing catalytic serine (Ser(148)) suggesting a selective carbamylation of the enzyme by AM6701 or WWL70 and confirming an essential role of this residue in catalysis. The ability to produce substantial quantities of functional, pure hABHD6 will aid in the downstream structural characterization, and development of potent, selective inhibitors.

Human monoacylglycerol lipase (hMAGL) plays a key role in homeostatic tuning of the endocannabinoid signaling system and supports aggressive tumorogenesis, making this enzyme a promising therapeutic target. hMAGL features a membrane-associated lid domain that regulates entry of endocannabinoid lipid substrates into the hydrophobic channel accessing the active site, likely from the membrane bilayer. The present work applied simultaneous surface plasmon resonance and electrochemical impedance spectroscopy measurements to show that, in absence of the substrate, hMAGL can remove phospholipid molecules from the membrane and, thereby, disintegrate pre-formed, intact, tethered phospholipid bilayer membrane mimetics (tBLMs) composed of unsaturated phosphatidylcholines. To probe the mechanism of hMAGL-induced on tBLMs compromise, we investigated the effect of wild type and mutant hMAGLs and hMAGL rendered catalytically inactive, as a function of concentration and in the presence of chemically distinct active-site inhibitors. Our data show that hMAGL's lid domain and hydrophobic substrate-binding pocket play important roles in hMAGL-induced bilayer lipid mobilization, whereas hydrolytic activity of the enzyme does not appear to be a factor.

Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger 2-arachidonoylglycerol. Agents that carbamylate hMGLs catalytic Ser(122) constitute a leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGL's conformational responses to two potent carbamylating inhibitors, AM6580 (irreversible) and AM6701 (slowly reversible). A dynamic, solvent-exposed lid domain is characteristic of hMGL's solution conformation. Both hMGL inhibitors restricted backbone enzyme motility in the active-site region and increased substrate binding-pocket solvent exposure. Covalent reaction of AM6580 with hMGL generates a bulkier carbamylated Ser(122) residue as compared to the more discrete Ser(122) modification by AM6701, a difference reflected in AM6580's more pronounced effect upon hMGL conformation. We demonstrate that structurally distinct carbamylating hMGL inhibitors generate particular conformational ensembles characterized by region-specific hMGL dynamics. By demonstrating the distinctive influences of two hMGL inhibitors on enzyme conformation, this study furthers our understanding at the molecular level of the dynamic features of hMGL interaction with small-molecule ligands.

The membrane-associated serine hydrolase, monoacylglycerol lipase (MGL), is a well-recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGL's membrane association upon its structure-function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge-neutral phospholipid bilayer nanodiscs enhanced hMGL's kinetic properties [apparent maximum velocity (Vmax ) and substrate affinity (Km )]. Hydrogen exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL-nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid-domain helix alpha4 and neighboring helix alpha6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzyme's substrate-binding channel. Covalent modification of membrane-associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane-associated topology of active and inhibited, covalently-modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzyme's conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity.

In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu(169);Leu(176))-to-Ser(Ser(169);Ser(176)) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser(122) with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser(122) residue as compared to that of Cys(122) in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys(122) residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser(122), and the mechanisms underlying hMGL inhibition by different classes of small molecules.