We statement a 2.75-? crystal structure of h/rFAAH complexed with an inhibitor, gene prospects to elevated endogenous levels of fatty acid amides and a range of behavioral effects that include analgesia (7C12), anxiolytic (8, 13, 14), antidepressant (13, 15), sleep-enhancing (16), and antiinflammatory (17C19) phenotypes. sleep-enhancing (16), and antiinflammatory (17C19) phenotypes. Importantly, these behavioral phenotypes happen in the absence of alterations in motility, weight gain, or body temperature that are typically observed with direct cannabinoid receptor 1 (CB1) agonists. Inhibition of FAAH therefore may offer a good way to produce the therapeutically beneficial phenotypes of activating the endocannabinoid system without the undesirable side effects that are observed with direct CB1 agonists. FAAH is definitely a member of a large class of enzymes termed the amidase signature class (20). These enzymes, which span all kingdoms of existence, use an unusual SerCSerCLys catalytic triad (21, 22) to hydrolyze amide bonds on a wide range of small-molecule substrates. Despite their atypical catalytic mechanism, amidase signature enzymes are inactivated by general classes of serine hydrolase inhibitors [e.g., trifluoromethyl ketones (23, 24), fluorophosphonates (25), -ketoheterocycles (26), carbamates (8, 27)]. First-generation FAAH inhibitors, such as methyl arachidonyl fluorophosphonate (MAFP) (25), were substrate-derived in structure and therefore lack selectivity for FAAH relative to additional lipid hydrolases. More recently, FAAH inhibitors with greatly improved selectivity have been explained (24, 26, 28). However, the mechanism by which these inhibitors accomplish potency and selectivity for FAAH remains unfamiliar, due in large part to a dearth of structural info on enzymeCinhibitor complexes. Indeed, to date, only a single crystal structure of FAAH has been reported, a complex between the rat enzyme (rFAAH) and MAFP (22). Attempts to date to accomplish structural information within the human being FAAH (hFAAH) protein have been hampered by low-expression yields in recombinant systems and Tal1 problematic biochemical properties (i.e., instability, aggregation). Here, we describe an alternative strategy that involves the mutagenic interconversion of the rat and hFAAH active sites. Specifically, we have manufactured a humanized rat (h/r) FAAH that contains a complete human being active site within the parent CNX-774 rat protein. This h/rFAAH exhibits the inhibitor level of sensitivity profile of hFAAH while keeping the high-recombinant manifestation yields and biochemical properties of the rat enzyme. We exploit these unique features to solve the crystal structure of h/r FAAH in complex having a selective small-molecule inhibitor. This CNX-774 structure reveals how inhibitors accomplish potency and specificity for hFAAH, therefore offering important insights to guide long term drug design attempts. Results Engineering a Humanized Form of Rat FAAH. We have previously reported an expression system to produce purified, active rFAAH protein bearing a His6 affinity tag in place of the N-terminal transmembrane website of the enzyme (29). This recombinant protein was used to determine the crystal structure of FAAH in complex with the general serine hydrolase inhibitor MAFP (22). hFAAH, despite posting 82% sequence identity with rFAAH, offers proven more difficult to express and purify. There are only a few reports within the recombinant manifestation of hFAAH using baculovirusCinsect cell (30, 31) and bacterial (30) systems; however, in these cases, hFAAH manifestation levels were not reported. Our own attempts to optimize fully the recombinant manifestation of FAAH proteins in have resulted in a robust protocol to generate high yields of rFAAH (20 mg of purified enzyme per liter of tradition). Although this protocol also produced a modest quantity of hFAAH (1 mg of purified protein per liter of tradition), this protein was much less stable and more prone to aggregation CNX-774 than rFAAH. As an alternative strategy, we wanted to create a humanized version of rFAAH, where the active site of the protein was converted to match the human being enzyme. Active-site residues were identified based on the crystal structure of rFAAH (22), and sequence comparisons recognized six of these amino acids that differed between rFAAH (L192, F194, A377, S435, I491, and V495) and hFAAH (F192, Y194, T377, N435, V491, and M495) [assisting info (SI) Fig. S1]. In the rFAAH structure, all of these residues except S435 interact with the arachidonyl chain of the bound methyl arachidonyl phosphonate (MAP), the MAFP inhibitor adduct. S435 was found to be in close proximity to, but not directly contacting, the arachidonyl chain. We mutated each of these 6 aa in rFAAH to the related residues in hFAAH, generating a h/rFAAH protein that indicated at levels much like rFAAH in (10 mg of purified protein per liter.