2-Arachidonoylglycerol plays a major role in endocannabinoid signaling, and is tightly regulated by the monoacylglycerol lipase (MAGL). Here we report the crystal structure of human MAGL. The protein crystallizes as a dimer, and despite structural homologies to haloperoxidases and esterases, it distinguishes itself by a wide and hydrophobic access to the catalytic site. An apolar helix covering the active site also gives structural insight into the amphitropic character of MAGL, and likely explains how MAGL interacts with membranes to recruit its substrate. Docking of 2-arachidonoylglycerol highlights a hydrophobic and a hydrophilic cavity that accommodate the lipid into the catalytic site. Moreover, we identified Cys201 as the crucial residue in MAGL inhibition by N-arachidonylmaleimide, a sulfhydryl-reactive compound. Beside the advance in the knowledge of endocannabinoids degradation routes, the structure of MAGL paves the way for future medicinal chemistry works aimed at the design of new drugs exploiting 2-arachidonoylglycerol transmission.

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Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gramnegative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn 2+ alone. The crystal structure of ZneB to 2.8 Å resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the β-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release.Cupriavidus metallidurans CH34 | heavy-metal resistance | resistance nodulation cell division | periplasmic adaptor protein M icroorganisms depend on protective mechanisms to survive the effects of toxic compounds in the environment. In Gramnegative bacteria, resistance nodulation cell division (RND) -driven efflux systems confer resistance to many drugs and heavy metals (1, 2). The canonical RND-based system is formed by the association of three components: one integral to the plasma membrane, one integral to the outer membrane, and a periplasmic connector. The plasma membrane protein (RND) is a substrate/ proton antiporter. The outer-membrane factor (OMF) spans a large part of the periplasm and provides the exit portal. The periplasmic membrane fusion protein (MFP) links these two components together. Based on the nature of their substrate, tripartite RND-driven efflux systems are divided into two subclasses: the hydrophobe/amphiphile efflux (HAE) and the heavy-metal efflux (HME) subclasses. The selectivity and function of many HAE-RND systems such as the Acr and Mex families have been determined, alongside crystal structures of the RND, OMF, and MFP components of various systems (3-13). However, knowledge of HME-RND systems, including substrate specificity and the basis for selectivity, is much more limited.The integral plasma membrane components of the RND-based efflux systems are thought of as the pump; however, the discovery of increasing functions of MFPs shows that these proteins also play a major role in substrate transport. Several MFPs bind their respective substrates (14-16), facilitate substrate transport (17, 18), and are essential for transport in vitro (17). MFPs are also involved in OMF recruitment (19), and are also found in Grampositive bacteria, where no OMF is pr...

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.

Monoacylglycerol lipase (MAGL) is a key enzyme responsible for the termination of endocannabinoid signaling. Its crucial role in 2-arachidonoylglycerol (2-AG) metabolism, together with the numerous pharmacological properties mediated by this endocannabinoid, emphasize the interest in MAGL as therapeutic target, along with the need to design potent and selective inhibitors. Meanwhile, the complexity of 2-AG degradation pathways underscores the need to use a purified source of enzyme in evaluation studies of new inhibitors. We report here the first heterologous expression and purification of human MAGL. A highly pure protein was obtained and allowed us to measure the affinity of several MAGL inhibitors for the human enzyme. Importantly, disulfiram (tetraethylthiuram disulfide), a compound used to treat alcoholism, and other disulfide-containing compounds were shown to inhibit MAGL with good potency, likely through an interaction with cysteine residues.