ABHD17A-membrane interaction is predicted to occur through two distinct domains. (A) Mapping of full-length AA ABHD17A to CG MARTINI3.0 model. (B) Representative mechanism for the binding of ABHD17A (red) to a PM-like membrane (1-palmitoyl-2-oleoylphosphatidylcholine [POPC]: light yellow, 1-palmitoyl-2-oleoylphosphatidylserine [POPS]: orange, cholesterol [CHOL]: yellow) in CG simulations: the protein, initially randomly oriented at a minimum distance of 5 nm from the bilayer, interacts with the membrane via both its N-terminal helix and the adjacent loop. (C) Conservation of the loop residues in the three ABHD17 isoforms in metazoans as shown with Weblogo (Crooks et al., 2004). (D and E) Membrane interaction of the N-terminal helix and conserved loop shown in the contacts analysis (D) and the occupancy maps (E). (F) Snapshot from a representative AA simulation, showing the back-mapped atomistic structure of ABHD17A with the addition of palmitoyl-tails to cysteine residues at its N-terminus. (G) Insertion depth analysis of AA simulations confirms that ABHD17A inserts into the membrane via both the palmitoylated N-terminus (residues 1–21) and the adjacent loop (residues 222–233). (H) Insertion depth analysis of ABHD17A 5C>S with AA simulations shows mutation of the five N-terminal cysteine residues to serine decreases both N-terminal and loop membrane insertion. (I) Restoring N-terminal hydrophobicity of the non-acylated ABHD17A (5C>W/L) rescues insertion depth of the N-terminus and loop in AA simulations. The data shown in G–I are averaged over four replicates, and the error bars indicate STDEV.