Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase

Abstract

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.

Figure 1

(a) The overall hMGL 3D structure (PDB ID:3HJU) showing the location of two conserved Trp-35 and Trp-289 residues relative to the active site of the enzyme. The lid domain is highlighted in magenta. (b) Fragment of this structure zoomed in to provide a view of the distal residues Trp-289 and Leu-232 that reside more than 18 Å away from the catalytic triad residues. (c) Close-up view of the identified remote site. Interactions between side-chains of Trp-289, Leu-232 and Arg-293 are highlighted.

Figure 2

(a) Real-time NMR spectra demonstrating spontaneous, sequential interconversions among different lid domain conformations in the transition pathway between two extreme conformations: open and closed (T = 310 K, pH 7.4). Variable temperature NMR experiments demonstrating (b) temperature effect on the rate of exchange between open conformations of sol-hMGL and (c) effect of temperature on the distribution of open-closed conformers for the H272A mutant (pH 7.4).

Figure 3

(a) Comparison of the far-UV CD spectra of sol-hMGL and mutant enzymes. The protein concentrations were ~10 µM. (b) Thermal denaturation curves of sol-hMGL and mutants. Enzymes were subjected to a temperature gradient in 20 mM sodium phosphate, 100 mM NaCl, 1 mM DTT buffer, pH 7.4, and unfolding was followed by monitoring the CD signal at 222 nm. Data points are shown in black circles and the sigmoidal fits used to determine Tm. (c) Folding of W289L mutant assessed by two-dimensional ¹H-¹⁵N HSQC NMR spectroscopy. Spectrum was acquired for uniformly ¹⁵N isotope-labeled sample at 310 K, pH 7.4, C = 150 µM.

Figure 4

(a) The downfield ¹H NMR spectra of sol-hMGL with temperature dependence behavior. The effect of single-point mutations and temperature for (b) W35A; (c) W289L; (d) W289F; (e) R293A and (f) L232G (pH 7.4).

Figure 5

Superposition of two-dimensional ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled proteins (a) open sol-hMGL (red) and W289F (blue), (b) open sol-hMGL(red) and W289L (blue), (c) closed sol-hMGL (red) and W289F (blue) and (d) closed sol-hMGL (red) and W289L (blue) at 300 K, pH 7.4.

Figure 6

Effect of hMGL binding with Compound-1 on the downfield resonances of (a) sol-hMGL; (b) W35A; (c) W289F; (d) R293A; (e) W289L and (f) L232G at pH 7.4, T = 310 K.

Figure 7

Plots of deuterium uptake vs time (30 s, 5 min, 15 min, 1 h and 4 h) for the 12 peptides (a–l) showing statistically different behavior for W289L mutant as compared to wt hMGL. Uncertainties (1σ), indicated by bars, reside inside the plotted symbol. (m) Differential deuterium uptake profiles of wt-hMGL and W289L at 4 h mapped onto the crystal structure of hMGL (PDB:3HJU). Increased differential deuterium uptakes are color-coded from yellow to red and reduced uptake is colored blue. Grey indicates regions where the deuterium uptake levels in the wt hMGL and W289L mutant are statistically the same. Grey, in addition, denotes regions where peptic peptides were not observed.

Figure 8

MD simulation results for wt hMGL and W289L mutant. Black dashed circles mark the substrate entrance area into the hMGL active site (binding pocket). (a) The wild type retains the open conformation throughput the 200 ns simulation. (b) W289L mutant moves towards the restricted conformation after 100 ns. Surface colors illustrate the electrostatic potential with red representing negative charges and blue representing positive charges. (c) Overlapping of W289L three-dimensional structures at 0 ns (red) and after 200 ns (blue) of MD simulation study. The C_α distance between the two representative residues Ser-155 and Gly-177 decreased 2-fold. The black arrows indicate the direction of the helix/loop movement.