Structures of the human peroxisomal fatty acid transporter ABCD1 in a lipid environment

Abstract

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to fatty acid catabolism and lipid biosynthesis. Its dysfunction underlies toxic cytosolic accumulation of VLCFAs, progressive demyelination, and neurological impairments including X-linked adrenoleukodystrophy (X-ALD). We present cryo-EM structures of ABCD1 in phospholipid nanodiscs in a nucleotide bound conformation open to the peroxisomal lumen and an inward facing conformation open to the cytosol at up to 3.5 Å resolution, revealing details of its transmembrane cavity and ATP dependent conformational spectrum. We identify features distinguishing ABCD1 from its closest homologs and show that coenzyme A (CoA) esters of VLCFAs modulate ABCD1 activity in a species dependent manner. Our data suggest a transport mechanism where the CoA moieties of VLCFA-CoAs enter the hydrophilic transmembrane domain while the acyl chains extend out into the surrounding membrane bilayer. The structures help rationalize disease causing mutations and may aid ABCD1 targeted structure-based drug design.

Fig. 1. Structural and Functional characterization of outward open human ABCD1.

a ATPase activity of ABCD1 reconstituted in liposomes or nanodiscs as a function of ATP concentration, n = 3 and error bars represent standard deviation b ATPase activity of DDM/CHS detergent purified ABCD1 in the presence of CoA esters of the indicated varying acyl chain composition, normalized to ABCD1’s basal rate at left, n = 6 (100 µM) or 3 (1 mM) and error bars represent S.E.M. Statistical significance by unpaired, two-tailed t-test p-values of <0.05, 0.01, 0.001, 0.0001 are indicated by *, **, ***, ****, respectively. c−f Map and structure of human ABCD1 outward open (OO) homodimer viewed from the membrane plane, made using only OO particles. EM density (0.04 contour) and ribbons are colored red and slate for each ABCD1 monomer, black sphere for R280, and green and yellow for modeled lipid-like entities and bound nucleotides, respectively. e Peroxisomal lumen with TMDs removed for clarity and f peroxisomal lumen with intracellular gate circled (dashed red line) with NBDs removed for clarity. TMs are numbered g electrostatic surface representation of ABCD1 viewed as a slice from the peroxisomal lumen.

Fig. 2. Comparison of IO and OO conformations of ABCD1.

a EM density map (0.04 contour) of human ABCD1 IO (cytoplasm facing) conformation colored yellow and gray for two monomers with similarly colored ribbon representing the protein backbone at right. b All Cα-atom overlay of IO and OO structures of human ABCD1 (gold and blue ribbons, respectively) with TMs numbered and external helix (EH) shown for IO conformation, with far viewing plane clipped to 10 Å. c Superposition of individual TM pairs from IO and OO structures with TM4 disrupting P263 & G266. d Comparison of NBDs of IO and OO structures aligned using NBD1 as reference from the lumenal viewpoint. Arrows depict inter coupling helix 2 (CH2) distances between Y296 Cα atoms.

Fig. 3. Comparison of IO and OO TMD cavities of ABCD1.

a All Cα-atom overlay of IO and OO ABCD1 structures (gold and blue sticks, respectively) but showing intracellular gate and surrounding residues viewed from the peroxisome lumen (left) and membrane plane (right). Equivalent residues from the two halves are distinguished by a prime symbol (b) same as (a) showing residues comprising the peroxisomal gate. c Surface representation of OO and IO structures of ABCD1 with solvent-exposed cavities colored red. d Slice through electrostatic surfaces of OO and IO structures of ABCD1 focused on the TMD region.

Fig. 4. Analysis of TMD mutations in ABCD1 and proposed substrate interactions.

a ABCD1 TMD with Cɑ atoms of the most frequently mutated residues shown as spheres colored slate and pink for the two ABCD1 monomers and divided into two main clusters (dashed boxes 1 & 2). b Select ABCD1 disease-causing mutations mapped onto ABCD1 IO structure. c EM density (0.04 contour) of ABCD1 showing modeled lipid-like entities (green) observed near gap in the OO structure (blue), overlaid onto the IO structure (gold). Unmodeled lipid-like density is shown pink. d Hypothetical mechanism of ATP-dependent fatty-acyl-CoA (green) translocation across the peroxisomal membrane (gray) mediated by ABCD1.