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

Le Thi My Le,Janarjan Bhandari,James Robert Thompson,Phuoc Xuan Dang,Amer Alam

doi:10.1038/s42003-021-02970-w

Le Thi My Le, Janarjan Bhandari + Show 3 more

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https://doi.org/10.1038/s42003-021-02970-w

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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.

Highlights

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to fatty acid catabolism and lipid biosynthesis
We utilized a tetracyclineinducible stable cell line to produce human ABCD1 in human embryonic kidney (HEK) 293 cells and tested its ATPase activity in detergent and in liposomes and nanodiscs comprising a mixture of porcine brain polar lipids (BPL) and cholesterol (Chol) previously used to characterize several other human ABC transporters[32,33] (Fig. 1a and Supplementary Fig. 1a)
Consistent with previously reported studies[34], ATP hydrolysis rates for ABCD1 were in the range of 10 nmol min−1 mg−1 in liposomes and nanodiscs, similar to values reported by other groups[34,35], and followed Michaelis Menten kinetics with Michaelis constant (KM) values of about 0.3 mM ATP (Fig. 1a)

Summary

Introduction

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to fatty acid catabolism and lipid biosynthesis. To define the ABCD1 structural elements that enable ATP dependent and specific VLCFA transport at the molecular level, we determined its structures in nanodisc reconstituted form in both a nucleotide-bound outward open (OO) conformation open to the peroxisomal lumen, and an inward open (IO) conformation open to the cytosol. In conjunction with ATP-driven functional assessments, our data reveal the conformational landscape associated with the ATP-dependent transport cycle of ABCD1 and highlight key features of its TMD that offer insights into its potential substrate-binding mechanism. These structures allow us to pinpoint the location of the most frequently occurring disease-causing mutations in the ABCD1 TMD that will allow for the conceptualization of structurefunction hypotheses based on X-ALD patient mutations[31]. These structures open the door for more accurate structure-guided design of ABCD1 targeted small molecule therapeutics and computational studies of ABCD1 structure and function

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