Structural and functional insights of the human peroxisomal ABC transporter ALDP

  1. Yutian Jia
  2. Yanming Zhang
  3. Wenhao Wang
  4. Jianlin Lei
  5. Zhengxin Ying
  6. Guanghui Yang

  • Curated by eLife

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    Evaluation Summary:

    This paper will be of interest to the lipid metabolism and transporter communities. Fatty acids that are too long to be transported into mitochondria are instead transported into peroxisomes for their break down i.e., beta-oxidation. The authors have determined the cryo-EM structure of human ABC transporter ABCD1 (ALDP), which translocates very-long-chain fatty acids (VLCFA) conjugated to coenzyme A across peroxisomal membranes, in complex with its substrate. While the work is well done, it is unclear what new mechanistic insights are gained from the ALDP structure. Also, the proposed conformational differences based on AlphaFold models should be taken with caution.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

Adrenoleukodystrophy protein (ALDP) is responsible for the transport of very-long-chain fatty acids (VLCFAs) and corresponding CoA-esters across the peroxisomal membrane. Dysfunction of ALDP leads to peroxisomal metabolic disorder exemplified by X-linked adrenoleukodystrophy (ALD). Hundreds of ALD-causing mutations have been identified on ALDP. However, the pathogenic mechanisms of these mutations are restricted to clinical description due to limited structural and biochemical characterization. Here we report the cryo-electron microscopy structure of human ALDP with nominal resolution at 3.4 Å. ALDP exhibits a cytosolic-facing conformation. Compared to other lipid ATP-binding cassette transporters, ALDP has two substrate binding cavities formed by the transmembrane domains. Such structural organization may be suitable for the coordination of VLCFAs. Based on the structure, we performed integrative analysis of the cellular trafficking, protein thermostability, ATP hydrolysis, and the transport activity of representative mutations. These results provide a framework for understanding the working mechanism of ALDP and pathogenic roles of disease-associated mutations.

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  1. Version published to 10.7554/elife.75039 on eLife
  2. eLife

    Evaluation Summary:

    This paper will be of interest to the lipid metabolism and transporter communities. Fatty acids that are too long to be transported into mitochondria are instead transported into peroxisomes for their break down i.e., beta-oxidation. The authors have determined the cryo-EM structure of human ABC transporter ABCD1 (ALDP), which translocates very-long-chain fatty acids (VLCFA) conjugated to coenzyme A across peroxisomal membranes, in complex with its substrate. While the work is well done, it is unclear what new mechanistic insights are gained from the ALDP structure. Also, the proposed conformational differences based on AlphaFold models should be taken with caution.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Fatty acids that are too long to be transported into mitochondria are instead transported into peroxisomes for their break down i.e., beta-oxidation. The ABC transporter ABCD1 (ALDP) translocates very-long-chain fatty acids (VLCFA) conjugates to coenzyme A across peroxisomal membranes. Here, the authors have determined the cryo EM structure of human ALDP in complex with the substrate to reveal interesting differences to other ABC transporters. They have mapped ALDP-associated disease mutants, and further assessed their impact on transport by following the uptake of C22:0-CoA and C24:0-CoA into a mammalian cell line.

    Strengthens: The cryo EM structure of human ALDP is well resolved and the impact of disease-causing mutants by monitoring uptake of C22:0-CoA and C24:0-CoA using LC-MS/MS rather than ATP hydrolysis (as is typically done) is appreciated.

    Weakness: The proposed coordination of fatty acid CoA lacks the highly-positive charges and one would expect to require for the coordination of this negatively-charged compound. Indeed, a similar structure deposited in bioRxiv has a different location for the substrate that makes more chemical sense. The proposed conformational differences based on AlphaFold models should be taken with caution. The paper is difficult to follow in places.

  4. eLife

    Reviewer #2 (Public Review):

    The structures of human proteins provide an essential framework for addressing their biomedically functions, including their mechanism, therapeutic design, and understanding the molecular basis of genetic mutations. The adrenoleukodystrophy protein (ALDP), a member of the D sub-family of ATP-binding cassette transporters or ABCD1, participates in the transport of free very long-chain fatty acids and their CoA esters across the peroxisomal membrane. The biomedical relevance is highlighted by the identification of over 900 mutations on ALDP that can lead to the severe genetic disorder X-linked adrenoleukodystrophy.

    The manuscript by Jia et al describes the structure determination of ALDP in the inward-facing conformation by single particle cryo-EM at a nominal resolution of 3.4 Å. Highlights of this work include:
    -identification of a short helix at the peroxisomal side, which distinguishes ABCD1 from the other three members of the ABCD family
    -identification of an extended alpha-helix at the C-terminus of ALDP that forms a coiled-coil with the symmetry related sequence, although it was not possible to build an accurate model of this region
    -identification of two CoA esters by ALDP in the cavity formed by the two TMDs (Fig. 2)
    -mapping of pathogenic mutations of ALDP on the structure (Fig 4a)
    -results of a cell base transport assay to evaluate the influence of several ALD-associated mutations on substrate translocation and specificity (Fig 4B)

    For the reasons noted above, the structures of human ABC transporters are important advances for providing the XYZ values for the atomic coordinates. That said, given the explosion of structural information on ABC transporters (for example, of the ~50 human ABC transporters, structures have been reported for the human A1, A4, B1, B2, B3, B4, B6, B8, B10, B11, C7, D4, G2, G5, G8 transporters in addition to D1), it is a challenge to interpret new results in the context of existing knowledge about ABC transporters. In this regard, the functional implications of the ALDP structure paper are more incremental, based on the following considerations:
    1. the locations of the mutations (Fig 4a) and the classification of mutations discussed on page 6 could have been deduced without determining the ALDP structure.
    2. the transport assays provide an approach to connecting structure and function, but too little information is provided about the assays to make those connections. Of the ~900 mutants, why were the variants in figure 4B selected? Where are they located in the structure? What do the results of the transport assay mean? For example, is the reduction of C22:S-CoA in the cytosol for the G343V mutant due to impaired transport or to reduced expression, misfolding or some other cause?

  5. eLife

    Reviewer #3 (Public Review):

    The manuscript describes the cryo-EM structure of the human Adrenoleukodystrophy protein (ALDP) that transports very long-
    chain fatty acids (VLCFAs) across the peroxisomal membrane. Disease mutations have been associated with ALDP dysfunction. ALDP belongs to the ABCD1 family of ABC transporters. The overall structure resembles other members of the family; it is a homodimer that consists of 12 TM helices and 2 NBDs. In the TMD they observe density for lipid like molecules that they have assigned as Co-A esters.

    Based on the available mutations, they mapped them on their structure and tried to classify them. They further evaluated some of these mutations in whole cell transport assays.

    Overall, the structure provides a good framework to understand the molecular basis of diseases mutations.

  6. Version published to 10.1101/2021.09.24.461756v1 on bioRxiv