Abstract

Fatty Acid Transport Protein 2 (FATP2) couples fatty acid uptake to intracellular activation and is associated with pathological lipid accumulation in cancer and nonalcoholic fatty liver disease. Here, we present cryo-electron microscopy structures of human FATP2 across its reaction cycle. Our structures suggest that FATP2 recruits fatty acids directly from the membrane interface through a hydrophobic tunnel. Catalysis involves a ~130{degrees} rotation of the C-terminal domain, a transition trapped by the antihypertensive drugs isradipine and benidipine. Both drugs lock the enzyme in a thioester-forming state, but benidipine exhibits superior efficacy by extending a bulky moiety into the primary catalytic tunnel to sterically block substrate entry. Furthermore, we identify a product inhibition mechanism where excess acyl-CoA traps the enzyme, potentially limiting metabolic overload. These findings provide a structural framework for understanding vectorial fatty acid channeling and a scaffold for developing modulators of metabolic flux.