Endocannabinoid Interaction with Human FABP1: Impact of T94A Variant.

Using recombinant human wild-type fatty acid binding protein 1 (WT FABP1 T94T) and variant (FABP1 T94A) protein, fluorescence binding assays, and circular dichroism, it was shown for the first time that WT FABP1 and T94A variant each have a single, relatively hydrophobic site for binding fluorescent NBD-labeled analogues of N-arachidonoylethanolamide (i.e. NBD-AEA) and 2-arachidonoylglycerol (NBD-2-AG) with high affinity. Most native N-acylethanolamides (NAEs), but only one 2-monoacylglycerol (i.e. 2-arachidonoylglycerol, 2-AG) displaced WT FABP1-bound fluorescently labeled endocannabinoids(ECs). While T94A variant did not differ in affinity for AEA and most other NAE, it exhibited modestly higher affinity for OEA, as well as higher affinity for 2-AG. Binding of AEA and 2-AG altered WT FABP1’s secondary structure more extensively than any other previous ligand examined. The T94A variant without ligand was more susceptible to temperature-induced unfolding. While the T94A variant was much less sensitive to ligand (i.e. AEA, 2-AG)-induced conformational change, nevertheless binding of AEA and 2-AG significantly stabilized T94A structure to thermal unfolding. These data provide the first evidence that ECs not only bind to but also alter the secondary structure of the human FABP1-with the latter markedly impacted by the T94A substitution, a variant highly associated with hepatic accumulation of lipids and non-alcoholic fatty liver disease (NAFLD). Importantly, NAFLD has been associated with elevated hepatic levels of ECs and FABP1.