Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency: A ROLE FOR GLOBAL CONFORMATIONAL CHANGES

Abstract

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.

Keywords: catalytic efficiency; conformational change; lipase; nuclear magnetic resonance (NMR); serine protease; spectroscopy.

FIGURE 1.

A, comparison of ¹H NMR downfield resonances for sol-hMGL and specific mutations from which assignments were made (pH 7.4, T = 310 K). B, partial ¹H,¹⁵N HSQC spectrum showing resonances from histidine side chains in the sol-hMGL construct (pH 7.4, T = 310 K). C, the spectrum of the S122C mutant showing the effect of protonation on the His-269 H^δ1 resonance (pH7.4, T = 275 K). D, the structure of hMGL highlighting lid subdomain and the relative positions of the mutated residues.

FIGURE 2.

A–C, crystal structures showing hydrogen-bonded His-269 in the catalytic triad (Ser-122-His-269-Asp-239) of hMGL (3HJU) (A), His-49 involvement in the hydrogen bonding network within the oxyanion hole (Gly-50, Ala-51, Met-123) (3HJU) (B), and the His-54 residue in the open form (3HJU) and the closed form (3PE6) (C). In contrast to these x-ray structures, mutagenesis with NMR clearly demonstrated that His-54 is hydrogen-bonded to Asp-197 in the open form and is not bonded in the closed form. D, the His-103-His-75 residues located at the interface between strand β4 and helix α2 forming a bridge between these secondary structures (3PE6).

FIGURE 3.

A, comparison of downfield ¹H NMR resonances of the soluble S122C and S122C/H269A mutant (pH 7.4, T = 275 K). Effect of substitution of active site His-269 for alanine. B, comparison of ¹H NMR downfield resonances of sol-hMGL with resonances of soluble H103A and H75A mutants (pH 7.4, T = 310 K).

FIGURE 4.

Effect of pH on the downfield (A) and upfield (B) side chain resonances of hMGL (T = 310 K). The red arrows indicate the change of His-269 chemical shifts with increasing pH. Relative populations of the open and closed hMGL conformations were calculated from the areas of His-54 (C) and His-269 (D) resonances.

FIGURE 5.

A, superposition of the two-dimensional ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled sol-hMGL (pH 7.4, temperature = 300 K) in the active (red) and in the inactive (blue) conformations. B–D, superposition of the two-dimensional ¹H-¹³C HSQC spectra of ¹³C-labeled sol-hMGL in the active (red) and in the inactive (blue) conformations (aliphatic region) (B) and aromatic region (C). D, one-dimensional ¹H NMR downfield resonances of individual sol-hMGL conformers at pH 7.4 and temperature = 300 K.

FIGURE 6.

A, open hMGL conformation stabilized by a cation-π interaction between Arg-57 (arginine switch) and His-272 (PDB code 3HJU). B, closed conformation with Arg-57 flipped away from His-272 (PDB code 3PE6).

FIGURE 7.

A, effect of His-272 mutations on the population of conformers (pH 7.4, T = 310 K). B, effect of temperature on the open-closed equilibrium for the H272S mutant (pH 7.4).

FIGURE 8.

A, the effects of paraoxon on the downfield resonances of sol-hMGL and H272A (pH 7.4, T = 310 K). B, the effects of paraoxon on the downfield resonances of H54A.