Phosphatidylethanolamine binds to human perilipins via a hydrophobic cleft in their 4-helix bundle domain for lipid droplet targeting.

Perilipins (PLINs) are a family of proteins that coat the surface of lipid droplets (LDs), the cell's main storage sites for fats, to control their formation, growth, and breakdown. These proteins share a common structure: an N-terminal PAT domain for initial targeting, a central region of repeating helices that insert into the LD surface, and a C-terminal 4-helix bundle for stable anchoring. While the PAT domain binds diacylglycerol to promote LD formation at the endoplasmic reticulum, the conserved 4-helix bundle's lipid-sensing role has remained elusive. Here, we show that this bundle contains a hydrophobic cleft that specifically binds phosphatidylethanolamine (PE), a cone-shaped lipid promoting membrane bending during LD budding, as predicted by AlphaFold3 (alphafoldserver.com) models and confirmed by docking simulations. Binding assays reveal that the isolated bundle strongly attaches to LD-like particles enriched in PE, but mutations closing the cleft block this interaction. In yeast cells, limiting PE reduces PLIN3 localization to LDs, an effect aggravated by cleft mutations but independent of LD size or number. This PE-binding ability is shared by PLIN2, PLIN4, and PLIN5 but missing in PLIN1, matching their structural differences. Overall, our work reveals how the 4-helix bundle lets PLINs detect and adapt to LD surface lipid makeup, explaining their varied cellular roles and opening paths for treatments in fat-storage diseases such as liver steatosis.