Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

Abstract

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency

“…This differs from x-ray structures of the open form where His-54 is shown as the N ␦1 -H tautomer and does not display this hydrogen-bonding (4,5), whereas the structure of the closed form indicated His-54 is in the more common N ⑀2 OH tautomer and is hydrogen-bonded to (Fig. 2C) (6). This difference may be explained by common difficulties in deriving the exact conformational state of histidine side chains from deposited x-ray structures.…”

“…To avoid the need for detergents and obviate enzyme aggregation/precipitation, a DNA construct expressing a soluble hMGL variant with two leucines substituted by two serines in the lid subdomain (double mutant L169S,L176S ) (solhMGL) was expressed and used in this study (6 (Fig. 1A).…”

“…1) with a significant loss of catalytic efficiency ( Table 1). The His-54 residue of hMGL is located at a substantial distance from the catalytic triad (4,6) such that this mutation could not have had a direct effect on His-269. Among the His-272 mutants, the H272S mutation in sol-hMGL most compromised the enzyme's catalytic efficiency, consistent with the conformational transition to the inactive form reflected by the absence of the His-269 and His-54 resonances in the down- field region (Fig.…”

“…In addition, the hydrogen bond between the His-54 -Asp-197 residues occurs between the lid subdomain and the core of the enzyme, and this may also be impacted by the conformational rearrangement of Arg-57 in this region of the enzyme (Fig. 2C) (5,6).…”

“…Published crystal structures of modified/ liganded forms of hMGL (4 -6) indicate that the enzyme has a typical lipase structure that includes a lid subdomain (residues 151-225) that can assume open or closed states and control, through its gating dynamics, substrate access to the active site (7)(8)(9)(10)(11)(12)(13). The open state has been observed in the absence and presence of bound ligand (4,5), whereas the hMGL closed conformation has only been reported in complex with a reversible inhibitor (6). Interspecies conservation of the overall hMGL lid architecture and dynamics has been suggested from x-ray studies and molecular dynamics simulations of bacterial MGL (14 -16).…”