Molecular structures of the eukaryotic retinal importer ABCA4
Abstract
The ATP-binding cassette (ABC) transporter family contains thousands of members with diverse functions. Movement of the substrate, powered by ATP hydrolysis, can be outward (export) or inward (import). ABCA4 is a eukaryotic importer transporting retinal to the cytosol to enter the visual cycle. It also removes toxic retinoids from the disc lumen to the cytosol. Mutations in ABCA4 cause impaired vision or blindness. Despite decades of clinical, biochemical, and animal model studies, the molecular mechanism of ABCA4 is unknown. Here we report the structures of human ABCA4 in two conformations. In the absence of ATP, ABCA4 adopts an outward-facing conformation, poised to recruit substrate. The presence of ATP induces large conformational changes that could lead to substrate release. These structures provide a molecular basis to understand many disease-causing mutations and a rational guide for new experiments to uncover how ABCA4 recruits, flips, and releases retinoids.
Data availability
The cryo-EM maps are deposited in the Electron Microscopy Data Bank (EMDB) under accession codes: EMD-23409, EMD-23410. The corresponding atomic models are deposited in the Protein Data Bank (PDB) under accession codes 7LKP and 7LKZ.
Article and author information
Author details
Funding
Howard Hughes Medical Institute
- Jue Chen
Helen Hay Whitney Foundation
- James Lee
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- David Drew, Stockholm University, Sweden
Publication history
- Received: September 28, 2020
- Accepted: February 18, 2021
- Accepted Manuscript published: February 19, 2021 (version 1)
- Version of Record published: March 4, 2021 (version 2)
Copyright
© 2021, Liu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,594
- Page views
-
- 533
- Downloads
-
- 11
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Structural Biology and Molecular Biophysics
Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC’s external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.
-
- Cell Biology
- Structural Biology and Molecular Biophysics
Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1’s lumenal domain. Phosphorylation of IRE1’s kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1’s RNase activity.