Molecular mechanism of activation of the immunoregulatory amidase NAAA

Significance There is a strong need for new analgesic and antiinflammatory medicines that are both effective and safe. Animal studies have shown that inhibition of N-acylethanolamine acid amidase (NAAA)—an intracellular enzyme that degrades the lipid mediator palmitoylethanolamide—causes profound analgesic and antiinflammatory effects. To facilitate the discovery of drugs targeting this protein and to better understand its mechanism of action, we determined its 3D structure. Our results illustrate the sequential steps leading to the activation of NAAA at lipid membranes, and reveal how current inhibitors block this enzyme. Palmitoylethanolamide is a bioactive lipid that strongly alleviates pain and inflammation in animal models and in humans. Its signaling activity is terminated through degradation by N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase expressed at high levels in immune cells. Pharmacological inhibitors of NAAA activity exert profound analgesic and antiinflammatory effects in rodent models, pointing to this protein as a potential target for therapeutic drug discovery. To facilitate these efforts and to better understand the molecular mechanism of action of NAAA, we determined crystal structures of this enzyme in various activation states and in complex with several ligands, including both a covalent and a reversible inhibitor. Self-proteolysis exposes the otherwise buried active site of NAAA to allow catalysis. Formation of a stable substrate- or inhibitor-binding site appears to be conformationally coupled to the interaction of a pair of hydrophobic helices in the enzyme with lipid membranes, resulting in the creation of a linear hydrophobic cavity near the active site that accommodates the ligand’s acyl chain.