Structure and activation mechanism of the BBSome membrane protein trafficking complex
- Harvard Medical School, United States
Abstract
Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a key regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the b-propeller domain of BBS1, the subunit most frequently implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis results from folding defects and the disruption of autoinhibition and activation.
Data availability
The EM density map for the BBSome has been deposited under accession code EMD-21144 and the EM density map for the BBSome:ARL6:GTP complex has been deposited under accession code EMD-21145. Masks and maps from multibody refinement are included as additional maps in these depositions. The corresponding atomic models have been deposited under accession codes 6VBU and 6VBV.
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Structure of the bovine BBSome (map)Electron Microscopy Data Bank, EMD-21144.
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Structure of the bovine BBSome:ARL6:GTP complex (map)Electron Microscopy Data Bank, EMD-21145.
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Structure of the bovine BBSome:ARL6:GTP complex (model)Protein Data Bank, 6VBV.
Article and author information
Author details
Funding
Pew Charitable Trusts
- Alan Brown
Internal Retinal Research Foundation
- Alan Brown
E. Matilda Ziegler Foundation for the Blind
- Alan Brown
Richard and Susan Smith Family Foundation
- Alan Brown
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Singh 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.
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Structure and activation mechanism of the BBSome membrane protein trafficking complex
eLife 9:e53322.
https://doi.org/10.7554/eLife.53322
Further reading
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- Structural Biology and Molecular Biophysics
The structures of the bovine and human BBSome reveal that a conformational change is required to recruit the complex to the ciliary membrane.
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- Structural Biology and Molecular Biophysics
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous and synthetic ligands, including covalent antagonist inhibitors GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent inhibitors weaken—but do not prevent—the binding of other ligands via an allosteric mechanism, rather than direct ligand clashing, by shifting the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with orthosteric ligand binding. Crystal structures reveal different cobinding mechanisms including alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent inhibitor binding pose. Our findings highlight the significant flexibility of the PPARγ orthosteric pocket, its ability to accommodate multiple ligands, and demonstrate that GW9662 and T0070907 should not be used as chemical tools to inhibit ligand binding to PPARγ.
