Structural basis of substrate recognition and translocation by human very long-chain fatty acid transporter ABCD1

Abstract

Human ABC transporter ABCD1 transports very long-chain fatty acids from cytosol to peroxisome for β-oxidation, dysfunction of which usually causes the X-linked adrenoleukodystrophy (X-ALD). Here, we report three cryogenic electron microscopy structures of ABCD1: the apo-form, substrate- and ATP-bound forms. Distinct from what was seen in the previously reported ABC transporters, the two symmetric molecules of behenoyl coenzyme A (C22:0-CoA) cooperatively bind to the transmembrane domains (TMDs). For each C22:0-CoA, the hydrophilic 3'-phospho-ADP moiety of CoA portion inserts into one TMD, with the succeeding pantothenate and cysteamine moiety crossing the inter-domain cavity, whereas the hydrophobic fatty acyl chain extends to the opposite TMD. Structural analysis combined with biochemical assays illustrates snapshots of ABCD1-mediated substrate transport cycle. It advances our understanding on the selective oxidation of fatty acids and molecular pathology of X-ALD.

Fig. 1. Substrate-stimulated ATPase activity assays and structure determination of ABCD1.

a ATPase activity of chABCD1 in the presence or absence of C22:0-CoA in detergent of LMNG + CHS. 0.05% methyl-β-cyclodextrin (M-β-CD) which functions as the solvent of substrate, was also tested as the control group. The data points were fitted with a Michaelis-Menten equation. b Substrate concentration-dependent ATPase activity of chABCD1 in detergent of LMNG + CHS and 2 mM ATP upon addition of varying fatty acyl-CoA. The data points were fitted with a Hill equation. All data points for a and b represent means of three independent measurements (n = 3) in detergent of LMNG and CHS. Error bars indicate standard deviation. Source data are provided as a Source Data file. c Refined cryo-EM map of three ABCD1 structures. The unsharpened maps are displayed as the outline to show the position of the detergent micelle. The cryo-EM map are colored by UCSF ChimeraX 1.2.5 according to the local resolution of the cryo-EM estimated by RELION 3.1 or cryoSPARC 3.1.

Fig. 2. Overall structure of apo-form ABCD1.

a Cartoon representation of the apo-form ABCD1. Two subunits are colored in green and rose, respectively. Transmembrane helices (TMs), C-terminal helices and coupling helices (CH1’ and CH2’) are labeled. Two extra lipid-like densities are shown as the blue mesh. The peroxisome membrane is indicated as the gray lines. b Top view of ABCD1 (from the inside of peroxisome). The six TMs of each subunit are numbered. c The ATPase activity of the C-terminal helix truncated variant (ΔGlu694-Thr745) and T693M mutant in detergent of LMNG + CHS and 2 mM ATP upon addition of C22:0-CoA at various concentrations. Each data point is the average of three independent experiments (n = 3), and error bars represent the means ± SD. The data points were fitted with a Hill equation. Source data are provided as a Source Data file.

Fig. 3. Structure of substrate-bound ABCD1 and the substrate binding pocket.

a Side and b Top view of the overall structure of C22:0-CoA-bound ABCD1. Two subunits of ABCD1 are colored in green and rose, respectively. The peroxisome membrane is indicated as the gray lines. The α-helices (termed α5a/5a’) in the peroxisome matrix is labeled. The two C22:0-CoA molecules are shown as yellow spheres, and the two cholesteryl hemisuccinate (CHS) molecules are shown as blue sticks. c Superposition of the apo-form (gray) against C22:0-CoA-bound ABCD1 (pink). TM4a/4a′ and TM4b/4b′ are labeled. d The C22:0-CoA molecule binding to TMDs. The density map of C22:0 and CHS, shown as blue mesh, are contoured at 5σ. The CHS and C22:0-CoA are shown as blue and yellow sticks, respectively. e The binding pocket of the CoA portion of C22:0-CoA. The binding residues are shown as sticks, and hydrogen bonds (≤3.5 Å) and salt bridges (≤4.0 Å) are shown as black dotted lines. The electrostatic surface properties of the binding pocket are color-coded by electrostatic potential generated by PyMOL 2.5.2. f The binding pocket of fatty acyl chain of C22:0-CoA. The hydrophobic residues surrounding the fatty acyl chain within 4.5 Å are shown as sticks. The fatty acyl chain and CHS are shown as blue and yellow sticks, respectively. g Relative ATPase activities of chABCD1 and mutants in detergent of LMNG + CHS and 2 mM ATP upon addition of C22:0-CoA. The relative activity represents the substrate-stimulated activity of chABCD1 or its mutant that harboring a single mutation of residues at the substrate-binding pocket. Each data point is the average of three independent experiments (n = 3), and error bars represent the means ± SD. One-way analysis of variance (One-way ANOVA) is used for the comparison of statistical significance of mutants and wild-type. The p value of R104A, R152A, K217A, K336A, Y337F, R401Q, A247W, G343V, P350W and A395W is 0.00019, 0.00062, 0.00076, 0.00092, 0.00053, 0.00063, 0.00014, 0.000178, 0.00031 and 0.00012. The p values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***. Source data are provided as a Source Data file.

Fig. 4. Structure of ATP-bound ABCD1 and comparison with the C22:0-CoA-bound structure.

a Cartoon representation of ATP-bound ABCD1. Two subunits of ABCD1 are colored in green and rose, respectively. ATP molecules are shown as yellow sticks, and Mg²⁺ ions are displayed as green spheres. The number of TMs are labeled. Conformational changes upon ATP binding accompanied with substrate release, as shown by b cutaway representation of the electrostatic surface, and c top view of C22:0-CoA-bound-ABCD1 and ATP-bound-ABCD1. TM3/TM3′ and TM4/TM4′ are shown as cartoon. The electrostatic surface was generated by PyMOL 2.5.2. The residue Arg280 is shown as sticks. Superposition of (d) the TMDs and (e) the CoA portion binding pocket of C22:0-CoA-bound ABCD1 (gray) against the ATP-bound form (rose). TM helices are shown as cartoons and numbered. The shifts of TM1/TM1′ and TM6/TM6′ are indicated in green or blue arrows, respectively. The residues interacting with CoA portion of C22:0-CoA are displayed as sticks.

Fig. 5. A working model for the ABCD1-mediated fatty acyl-CoA translocation.

Schematic illustration of the transport cycle of ABCD1, inferred from the three resolved structures. The apo-form ABCD1 adopts an inward-facing conformation, with the two NBDs properly separated via the C-terminal helical crossover (resting state). Cooperative substrate binding drags two TMDs approaching each other, resulting in a narrowed inward-facing transport cavity (substrate-bound state). Upon ATP binding, the NBDs approach each other and dimerize, making ABCD1 in an outward-facing conformation, and facilitating the substrate release into the peroxisome (post-translocation state). Finally, the hydrolysis of ATP resets ABCD1 to the resting state, and ready for another transport cycle. The CoA portion and fatty acyl chain of fatty acyl-CoA are shown as green ovals and brown curved lines, respectively.