Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets.

Giulia Palermo,Andrea Cavalli,Pablo Campomanes,Marco De Vivo,Inga Bauer,Andrea Armirotti,Ursula Rothlisberger,Stefania Girotto

doi:10.1371/journal.pcbi.1004231

Abstract

The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.

Highlights

Fatty acid amide hydrolase (FAAH—Fig 1) [1,2,3] and monoacylglycerol lipase (MAGL)[4] are two hydrolytic enzymes that mainly regulate the endocannabinoid system
Through the integration of microsecond-long molecular dynamics simulations with mutagenesis and kinetic experiments, our study suggests that structural flexibility, gating residues and multiple cavities in one catalytic site are keys to lipid selection in the endocannabinoid system
Our study suggested that anandamide assumes hydrolysis-prone conformations by moving its flexible arachidonoyl chain between the membrane access (MA) and acyl-chain binding (AB) cavities interceded by the dynamic paddle residues that act as a gate between these two binding cavities

Summary

Introduction

Fatty acid amide hydrolase (FAAH—Fig 1) [1,2,3] and monoacylglycerol lipase (MAGL)[4] are two hydrolytic enzymes that mainly regulate the endocannabinoid system These enzymes act on the endocannabinoid signaling mostly through hydrolysis of the endogenous substrates anandamide and 2-arachidonoylglycerol (2-AG), respectively.[5,6,7] Both FAAH and MAGL can hydrolyze other lipids less efficiently.[8,9,10] Regulation of the endocannabinoid system is a promising strategy for treating pain, cancer, and other inflammatory-related diseases, suggesting both FAAH and MAGL as effective drug targets.[11,12,13,14,15,16,17,18] It is crucial to decipher the mechanisms for substrate selection and catalysis, which might help in the rational design of new therapeutics that act by modulating the endocannabinoid system. Building on our own and other relevant studies on FAAH catalysis and inhibition,[19,20,21,22,23,24,25,26,27,28,29,30,31,32,33] we provide here an elucidation of the main structural and kinetic features involved in substrate selection during FAAH catalysis

Methods

Results

Discussion

Conclusion