SMALL ANGLE X-RAY SCATTERING STUDIES REVEAL IMPORTANT CLUES FOR MEMBRANE BINDING AND ACTIVITY OF FATTY ACID AMIDE HYDROLASE (FAAH)

Amides, esters, and ethers of long-chain polyunsaturated fatty acids, collectively referred to as “endocannabinoids”, represent a growing family of lipid signaling mediators found in several tissues with a wide variety of biological actions. The main members of this group of molecules are anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol. Fatty acid amide hydrolase (FAAH) is a typically membrane enzyme that catalyzes the conversion of AEA into arachidonic acid and ethanolamine, thus terminating the signaling of this endocannabinoid1. The homodimeric structure of FAAH has been deducted as a biological unit from the crystallographic structure of a mutant rat enzyme (TM-FAAH), a catalytically active form still able to bind the membranes even if missing the -helices mainly responsible for this interaction. Until now, direct information on FAAH aggregation state in solution and in the presence of inhibitors, substrate analogues, or lipids is still lacking.In this study we analyzed the oligomerization state of TM-FAAH in solution by small angle X-ray scattering (SAXS). We found that, among several effectors, high concentrations of Tris buffer (≥ 0.5 M) are able to stabilize monodisperse oligomers. Data analysis shows that these oligomeric structures have a value of radius of gyration of 129±13 Å, as calculated from the p(r) function (in accordance with the Guinier analysis) and a value of the maximum dimension of the particle (Dmax) of about 410±10 Å. Superimposing the crystallographic unit of FAAH (pdb entry 1MT5.pdb), which is an octamer of dimers, to the low resolution DAM model of our oligomers, we observed that these last are composed of three octamers revealing an unprecedented oligomerization state of FAAH in solution. Furthermore, we studied the structural effects of different FAAH inhibitors observing an increase of the oligomerization state of the protein in the presence of these molecules. Then we paralleled this structural information with a functional analysis of the enzyme investigating the role of these inhibitors in modulating the membrane association of FAAH using both fluorescence resonance energy transfer measurements (FRET) and Laurdan fluorescence. Taken together, the complementary information collected allowed to understand the molecular mechanisms leading to FAAH function. It also revealed the presence of an unprecedented oligomerization state of FAAH and a key role of inhibitors in favouring the subunit-subunit interactions that may impair the FAAH activity.1. Mei G. Di Venere A. Gasperi V. Nicolai E. Masuda K. R. Finazzi Agrò A. Cravatt B. F. and Maccarrone M. (2007) J. Biol. Chem. 282, 3829-3836.[...]