Membrane phospholipid bilayer as a determinant of monoacylglycerol lipase kinetic profile and conformational repertoire

Abstract

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 α4 and neighboring helix α6 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.

Figure 1

Localization of comformational changes in hMGL induced by phospholipid bilayer nanodiscs. (a) Deuterium incorporation curves derived from HX mass spectra for the peptides generated from hMGL pepsin digestion and designated by their amino acid residue numbers in 6-His-hMGL. Relative deuterium incorporation (Da) is plotted versus time of hMGL incubation in D₂O in the absence (blue lines, ) or presence (red lines, ) of POPC/POPG bilayer nanodiscs. The maximum amount of deuterium incorporation possible for each respective peptide is designated on the y-axis of each kinetic plot. Data were obtained through peptide level HX MS analysis of hMGL. The error of peptide HX MS mesurements with this experimental setup was ±0.50 Da, as determined by replicate analysis of peptide standards in prior HX MS work with this instrumentation., (b) The hMGL peptides for which deuterium uptake curves are presented in panel (a), above, are highlighted (orange) in the wild-type hMGL structure representation derived from PDB ID: 3JW8.²⁶ Residue designations in red type correspond to 6-His-hMGL.

Figure 2

Localization of conformational changes in hMGL induced through active-site Ser carbamylation by the covalent inhibitor, AM6580. (a) Structure of AM6580. (b) Deuterium incorporation curves derived from HX mass spectra for peptides generated from hMGL pepsin digestion and designated by their amino acid residue numbers in 6-His-hMGL. Relative deuterium incorporation (Da) is plotted versus time of hMGL incubation in D₂O in the presence of POPC/POPG phospholipid bilayer nanodiscs and without (red lines, ) or with (green lines, ) AM6580. The maximum amount of deuterium incorporation possible for each respective peptide is designated on the y-axis of each kinetic plot. Data were obtained through peptide-based HX MS analysis. The error of peptide HX MS mesurements with this experimental setup was ±0.50 Da, as determined by replicate analysis of peptide standards in prior HX MS work with this instrumentation., (c) Schematic depicting the docking of the AM6580-derived fluorenyl piprazine group covalently attached to hMGL active-site Ser¹²² (i.e., Ser¹²⁹ for 6-His-hMGL) in a portion of the hMGL structure representation derived from PDB ID: 3JW8.²⁶ Helix-α6 peptide 217–223 (orange) is shielded by the carbamylating-group modification at Ser¹²².

Figure 3

MD simulation of hMGL interaction with membrane phospholipid bilayer. Snapshots of hMGL (structure derived from PDB ID: 3JW8) with a phospholipid bilayer membrane of the same composition as in the experimental nanodiscs (POPC: POPG, 3:2 molar ratio) after 10 ns of MD simulation. The enzyme is depicted: (a) unliganded as apo-hMGL and (b) occupied with carbamylating inhibitor, AM6580, in the active site. Helix α4 in the lid domain and nearby helix alpha 6 are depicted in red, and AM6580 (b) is highlighted within the green oval and depicted in green.

Figure 4

Schematic superposition of the X-ray structure of hMGL (cyan, PDB ID: 3JW8) and the model of hMGL obtained after 10 ns of MD simulation at a water/phospholipid interface (gray). (a) Side view of hMGL; (b) top view of hMGL rotated 90° as indicated from the orientation in (a). The structures are virtually identical, except for helices α4 and α6 and the loop region from amino acid residues 177–192 connecting helices α4 and α5. The movements of helices α4 and α6 induced by a phospholipid bilayer (POPC:POPG, 3:2 molar ratio) are indicated by red arrows and red numerals specifying the extent of movement. Active site Ser¹²² (i.e., Ser¹²⁹ for 6-His-hMGL) is highlighted.