Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment
- Rensselaer Polytechnic Institute, United States
- Binghamton University, United States
- University of Vermont, United States
- Thomas Jefferson University, United States
Abstract
Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for many biological processes, including neuromuscular and brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Here for the first time, we show that a minimal Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules. Using nuclear magnetic resonance (NMR) titration and chemical exchange saturation transfer (CEST), mutagenesis and circular dichroism spectroscopy (CD), a Nup358 a-helix encompassing residues 2162-2184 was identified, which transitioned from a random coil to an a-helical conformation upon BicD2-binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 though a 'cargo recognition a-helix', a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility.
Data availability
Protein backbone assignments have been deposited in the BMRB under accession code 5182. All other data generated or analyzed during this study are included in the manuscript and supporting files; Source Data files have been provided for Figures 1, 2, 3, 4, 5, 6, 7, and 8.
Article and author information
Author details
Funding
NIH Office of the Director (R01 GM144578)
- M Yusuf Ali
- Sozanne R Solmaz
- Chunyu Wang
NIH Office of the Director (CA206592)
- Chunyu Wang
NIH Office of the Director (AG069039)
- Chunyu Wang
NIH Office of the Director (R15 GM128119)
- Sozanne R Solmaz
Chemistry Department and the Research Foundation of SUNY
- Sozanne R Solmaz
NIH Office of the Director (R35 GM136288)
- Kathleen M Trybus
NIH Office of the Director (R03 NS114115)
- M Yusuf Ali
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Gibson 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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Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment
eLife 11:e74714.
https://doi.org/10.7554/eLife.74714
Further reading
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Ciliary rootlets are striated bundles of filaments that connect the base of cilia to internal cellular structures. Rootlets are critical for the sensory and motile functions of cilia. However, the mechanisms underlying these functions remain unknown, in part due to a lack of structural information of rootlet organization. In this study, we obtain 3D reconstructions of membrane-associated and purified rootlets from mouse retina using cryo-electron tomography. We show that flexible protrusions on the rootlet surface, which emanate from the cross-striations, connect to intracellular membranes. In purified rootlets, the striations were classified into amorphous (A)-bands, associated with accumulations on the rootlet surface, and discrete (D)-bands corresponding to punctate lines of density that run through the rootlet. These striations connect a flexible network of longitudinal filaments. Subtomogram averaging suggests the filaments consist of two intertwined coiled coils. The rootlet’s filamentous architecture, with frequent membrane-connecting cross-striations, lends itself well for anchoring large membranes in the cell.
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Although the αC-β4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, local spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in this motif abolishes the synergistic high-affinity binding of ATP and a pseudo substrate inhibitor, we used LSP to interrogate the F100A mutant. This comparison highlights the importance of the αC-β4 loop and key residues at the interface between the N- and C-lobes. In addition, we delved more deeply into the structure of the apo C-subunit, which lacks ATP. While apo C-subunit showed no significant changes in backbone dynamics of the αC-β4 loop, we found significant differences in the side chain dynamics of K105. The LSP analysis suggests disruption of communication between the N- and C-lobes in the F100A mutant, which would be consistent with the structural changes predicted by the NMR spectroscopy.
