Structural And Functional Studies On The Human Very Long-chain Fatty Acyl-CoA Transporter ABCD1

Fatty acid is an important nutrient that plays an important role in the maintenance of cellular homeostasis.Fatty acids not only provide energy for vital biochemical processes through the fatty acid oxidation pathway,but also are involved in multiple physiological processes such as lipid synthesis,post-translational modification of proteins and cell signal transduction.However,excess fatty acids need to be degraded by the oxidation pathway in order to prevent their intracellular accumulation that can lead to the cell damages.In mammalian cells,the oxidation of short-chain,mediumlong-chain and long-chain fatty acids occurs in the mitochondria,while the oxidation of very long-chain fatty acids(VLCFAs),branched-chain fatty acids and bicarboxylix acids is performed in peroxisomes.Human adenosine triphosphate-binding cassette transporter subfamily D member 1(hABCD 1)which belongs to the ABCD subfamily of the ATP-binding casette(ABC)transporter superfamily is located on the peroxisomal membrane.ABCD1 transports coenzyme A esters of saturated or monounsaturated VLCFAs(fatty acyl-CoA)into the peroxisome matrix for β-oxidation against the concentration gradient by utilizing the energy generated by ATP hydrolysis.Dysfunction of ABCD1 prevents VLCFA from entering the peroxisome for β-oxidation,thus causing X-linked adrenoleukodystrophy(X-ALD),which is a rare recessive X-lingked disorder with a prevalence of up to 1/20,000 in males.However,the molecular basis of pathogenesis of X-ALD is still unclear due to the absence of the structures and molecular mechanism interpratation of ABCD1.In this project,by replacing the N-terminal 1-63 resdiues of hABCD1 with the Nterminal 1-65 residues of PMP-4,a homolog of hABCD 1 from Caenorhabditis elegans,we obtained a recombinant chimeric ABCD 1(termed chABCD1),which had a higher expression level,purity and homogeneity.ATPase activity assays indicated that chABCD1 displayed the same substrate specifity,and a higher substrate-stimulated ATPase activity compared to the full-length hABCD1.Using the single particle cryogenic electron microscope(Cryo-EM),we determined three structures of chABCD1:apo-form,behenoyl Coenzyme(C22:0-CoA)-and ATP-bound structures at the resolution of 3.5,3.6 and 2.8 (?),respectively.The apo-form chABCD1 adopts an inward-facing conformation,with two nucleotide binding domains(NBDs)separated and two transmembrane domains(TMDs)forming a V-shaped transport cavity open towards the cytosol.The C22:0CoA-bound ABCD1 structure also displays an inward-facing conformation,but two TMDs shift towards the center of translocation cavity,leading to the two NBDs approaching to each other.In the structure of C22:0-CoA-bound ABCD1,two C22:0CoA molecules display a unique binding pattern:for each C22:0-CoA molecule,the 3’phospho-ADP moiety of CoA portion inserts into the hydrophilic pocket of one TMD,and the succeeding pantothenate and cysteamine moieties cross the inter-domain cavity,whereas the hydrophobic fatty acyl chain extends to a hydrophobic pocket surrounded by another TMD and membrane.In the structure of ATP-bound ABCD1,ATP binding trigers the dimerization of the two NBDs,which results in an "outward-facing"conformation of ABCD1,making the cavity opening towards the peroxisomal matrix.Meanwhile,the rearrangements of the transmembrane helices disrupt the substrate binding pocket,and thus allow the substrate to be released into the peroxisomal matrix.Our structural analysis combined with biochemical assays shows the two symmetric molecules of behenoyl coenzyme A(C22:0-CoA)cooperatively bind to the transmembrane domains and clearly illustrates snapshots of ABCD1-mediated VLCFACoA recognition,translocation and release.It advances our understanding of the translocase mechanism of ABC transporters and provides a structural framework to the molecular pathologenesis of X-ALD caused by ABCD1 mutations.