Abstract
<jats:p>Fatty acid transport proteins (FATPs) are responsible for efficient uptake of fatty acids (FAs), essential biomolecules that play critical roles in development and growth. However, the lack of three-dimensional structures of FATPs has hindered our understanding of their mechanisms. Here, we report two cryo-EM structures of human FATP2a: wild-type (WT) FATP2a in intermediate state at 2.93 Å and a catalytic mutant of FATP2a in pre-catalytic state at 2.76 Å. In the two structures, both the oleoyl-AMP intermediate and ATP occupy the same central pocket with the acyl group of oleoyl-AMP pointing upwards and placed in a hydrophobic tunnel while the phosphate groups of ATP pointing downwards and interacting with a central loop. The structures also reveal that FAs from the membrane must undergo a two-step translocation to access the catalytic center. The binding of a new FA before the completion of a working cycle may be crucial to ensure the catalytic and transport efficiency. Furthermore, we demonstrate that FATP2a transport FAs, strictly dependent on the activation of FAs through the acyl-CoA synthetase activity. Our findings provide important insights into the working mechanism of FATPs and offer a structural basis for the development of inhibitors targeting FATPs to treat related diseases.</jats:p>