Multiscale Analysis of PNPLA2 and PNPLA3 Membrane Targeting

Lipid droplets (LDs) are dynamic organelles that regulate cellular lipid storage and mobilization through the coordinated action of LD-associated proteins. Patatin-like phospholipase domain-containing proteins PNPLA2 (ATGL) and PNPLA3 are central regulators of lipid metabolism, yet the molecular mechanisms underlying their membrane targeting and distinct enzymatic activities remain poorly understood. Here, we combine coarse-grained and all-atom molecular dynamics simulations with enhanced sampling to investigate how PNPLA2 and PNPLA3 associate with endoplasmic reticulum (ER) and LD membranes. Despite sharing a conserved N-terminal patatin domain, the two proteins exhibit distinct membrane-binding modes driven by divergent C-terminal amphipathic helices. In both proteins, membrane association is mediated primarily by deep insertion of C-terminal helices, while the patatin domain provides surface contact. PNPLA2 forms a deeply embedded U-shaped helical bundle on LDs that induce pronounced membrane curvature and promote opening of the catalytic dyad, consistent with its high triglyceride lipase activity. In contrast, PNPLA3 engages membranes through a more flexible helical arrangement that maintains a compact catalytic geometry and limits substrate accessibility. Membrane composition further modulates these interactions and leads to protein-specific lipid redistribution and curvature remodeling. Fluorescence microscopy experiments validate the computational predictions and demonstrate that mutation of a single arginine residue within the C-terminal region is sufficient to reduce LD targeting of both proteins. These results establish a mechanistic connection between membrane binding, conformational plasticity, and catalytic regulation in PNPLA2 and PNPLA3. Our work provides molecular insights into how lipid environments tune the function of LD-associated enzymes. Author SummaryLDs are essential cellular organelles that control how fats are stored and released, a process that relies on the precise recruitment and regulation of lipid-metabolizing enzymes. Our work focuses on two closely related enzymes, PNPLA2 (ATGL) and PNPLA3, which play central but distinct roles in lipid metabolism and metabolic diseases. Using a combination of multiscale modeling simulations and fluorescence microscopy, we examine how these proteins recognize and bind to ER and LD membranes. Although PNPLA2 and PNPLA3 share a conserved catalytic core, we show that they interact with membranes in different ways due to differences in their C-terminal amphipathic helices. We find that PNPLA2 forms a deeply embedded helical arrangement that reshapes the membrane and promotes access to its catalytic site, which explains why it typically shows strong lipase activity. In contrast, PNPLA3 adopts a more compact membrane-bound catalytic geometry that limits substrate access and enzymatic activity. We further applied fluorescence microscopy to experimentally validate the computational predictions. The results show that mutation of a single arginine residue within the membrane-binding helix reduces LD targeting. These findings reveal how membrane association and protein conformational dynamics jointly regulate catalytic accessibility and activity.