Structural Insights into the Broad Substrate Recognition Mechanism of Human ABCD3

Human peroxisomal ABC subfamily D transporters, ABCD1, ABCD2, and ABCD3, transport acyl-CoA molecules and bile acid precursors, and thereby participate in fatty acid metabolism and cholesterol homeostasis. ABCD3 can recognize a wide range of substrates, including polyunsaturated fatty acyl-CoAs, dicarboxylic acyl-CoAs, and ATP molecules. However, the structural mechanism of this broad substrate recognition by ABCD3 remains largely unknown. Here we report five cryo-electron microscopy structures of ABCD3: the apo state, the eicosapentaenoyl coenzyme A (C20:5-CoA)-bound state, the cholesteryl hemisuccinate (CHS)-bound state, the ATP-bound inward-facing state, and the ATP-bound outward-facing state. In the C20:5-CoA-bound state, the CoA moiety is recognized by ABCD3 in a similar manner to ABCD1, but the long, unsaturated bent acyl chain moiety is accommodated in a large cavity between protomers, distinct from the saturated straight acyl chain moiety-binding site observed in ABCD1. Furthermore, we found that the structurally and chemically different substrates, ATP and CHS, bind to distinct hydrophilic and hydrophobic sites, respectively, in the transmembrane domain of ABCD3. Combined with molecular dynamics simulations, these structures, encompassing both inward- and outward-facing conformations, reveal the molecular mechanism by which ABCD3 recognizes and transports a broad range of substrates.Main text