Amphipathic Helices Enable Fatty Acid Uptake and Activation by Human FATP2 at ER Contact Sites

Fatty acid transport proteins (FATPs) couple fatty acid uptake to metabolic activation; a process frequently dysregulated in metabolic disease and cancer. However, the structural basis of the coupling has remained unclear. Here we combine native mass spectrometry and cryo-EM to capture fatty acyl-adenylate intermediates and determine the first high-resolution structures of a FATP family member, FATP2. The structures reveal two functionally separable modules: an amphipathic helix module enabling membrane-dependent uptake and a catalytic activation module mediating metabolic trapping. Structure-guided mutagenesis demonstrates that the helices are essential for fatty acid uptake, whereas a catalytically inactive splice variant (FATP2b) uncouples membrane-dependent uptake from activation. Molecular dynamics simulations indicate that the two amphipathic helices induce lipid-packing defects and membrane thinning. Consistently, super-resolution live-cell imaging and co-immunoprecipitation show that FATP2 localizes to the endoplasmic reticulum and preferentially associates with ER–organelle contact sites. Together our data establish FATP2 as a prototype for membrane geometry-coupled metabolic enzymes and provide a structural framework for the development of FATP-targeted therapeutics.