During vision, the retinoid isomerase RPE65 transiently associates with cell membranes to process retinyl esters, yet how the membrane environment and protein acylation together organize this soluble enzyme are unresolved. Prior work proposed that RPE65 dimerizes on membranes and that a conserved palmitate could drive insertion, but the structural changes and their mechanistic basis remain undefined. Here, we show, using long atomistic simulations, that the membrane acts as a scaffold for RPE65, suppressing backbone fluctuations and tightening the dimer interface, establishing specific phosphatidylcholine contacts, including within the hydrophobic tunnel, and stabilizing the membrane-binding helix, with palmitoylation mainly increasing insertion depth without being required for helix insertion or sustained membrane association. Relative to solution, the membrane narrows residue-wise fluctuations and compresses inter-protomer spacing, with contacts centered at M285-G286, S307-S307, and R413 with P387/T389 becoming persistent on the bilayer but transient in solution. Time-averaged density maps resolve three phosphatidylcholine high-density regions at two membrane-facing motifs and inside the hydrophobic tunnel, indicating that endogenous lipids sample the putative retinyl ester substrate path rather than remaining excluded from it. Upon binding, the I115 to G125 segment remains extended and alpha helical, whereas in solution it collapses; across palmitoylated and unpalmitoylated proteins, helix stability follows insertion state rather than acylation. Our data support a general model in which the membrane is a primary stabilizer of soluble lipid enzymes, and palmitoylation enhances, rather than enables, their engagement.
Santos et al. (Sun,) studied this question.