Structure and Dynamics of Human Perilipin 3 Membrane Association

Lipid droplets (LDs) are a unique organelle that contain an oil core mainly composed of triglycerides that is surrounded by a phospholipid monolayer and coated by LD-associated proteins called perilipins (PLINs). There are five PLINs in human that generally contain a highly conserved N-terminal PAT domain of unclear function, followed by variable stretches of amphipathic helices previously implicated in LD association, and a C-terminal 4-helix bundle domain. During the process of LD biogenesis, perilipin 3 (PLIN3) is the first PLIN to be recruited to nascent LDs as they emerge from the endoplasmic reticulum. However, how PLIN3 recruitment to LDs is affected by lipid composition, and the structure and dynamics of PLIN membrane association has not been examined in detail. Here, using liposome co-sedimentation and LD-flotation assays we found that PLIN3 membrane association was enhanced by membrane packing defects and further promoted by the triglyceride precursors diacylglycerol (DAG) and phosphatidic acid. Hydrogen-deuterium exchange mass spectrometry experiments identified that both the PAT domain and amphipathic helices undergo a transition upon membrane binding from intrinsically disordered to an ordered state. Using constructs containing different portions of the PAT domain and amphipathic helices, we identify a new functional PAT domain that contains all conserved residues and is strongly recruited to DAG enriched membranes. Consistent with these findings, this newly determined functional PAT domain is predicted to form a triangular helical tertiary structure by alphafold and RoseTTAfold. Taken together, this study reveals molecular details of PLIN3 recruitment to membranes and suggests a role for the PAT domain as a major driver of PLIN3 to DAG enriched membranes, which is consistent with the localization of PLIN3 on early nascent LDs.