Allosteric ensembles elucidate mechanisms of inhibition in the human soluble epoxide hydrolase

Human soluble epoxide hydrolase (sEH) is an important modulator of cardiovascular, brain and renal health, and a promising therapeutical target for several pathologies. Its C-terminal domain (CTD) possesses hydrolytic activity which is responsible for decreasing the level of beneficial bioactive epoxy fatty acids. In this work we uncover the mechanisms of allosteric inhibition exerted on sEH CTD through the cysteine residues C522 and C423. In silico investigations coupled with biophysical and biochemical characterizations revealed that allosteric inhibition by the endogenous lipid mediator 15-deoxy-Δ^12,14-Prostaglandin J₂ (15d-PGJ₂) at C423 and C522 operates through distinct structural and dynamic mechanisms. Furthermore, the two allosteric sites C522 and C423 differ in their ability to generate an allosteric response upon binding of different effectors, in that association of divalent copper ions at C423 caused inhibition, whilst binding at C522 did not. The newly generated knowledge of the chemical properties of the effectors, their distal binding site and their specific allosteric signaling to functional sites will be instrumental to develop allostery-based therapeutics for sEH.