Coil-to-Helix Transition at the Nup358-BicD2 Interface Activates BicD2 for Dynein Recruitment

Abstract

Nup358, a nuclear pore protein, facilitates a nuclear positioning pathway that is essential for brain development. Nup358 binds and activates the auto-inhibited dynein adaptor Bicaudal D2 (BicD2), which in turn recruits and activates the dynein machinery to position the nucleus. The molecular details of the Nup358/BicD2 interaction remain poorly understood. Here, we show that a minimal dimerized Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules. Using nuclear magnetic resonance (NMR) titration and chemical exchange saturation transfer (CEST), a Nup358-helix encompassing residues 2162-2184 was identified, which transitioned from random coil to an alpha-helix 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 the cargo adaptor Nup358 though a 'cargo recognition alpha-helix', a structural feature that may stabilize BicD2 in its activated state, promoting activation of dynein motility.

In vitrocharacterization of the full-length human dynein-1 cargo adaptor BicD2

“…We characterized the BicD2 reactivity with its known cargo protein RanBP2. There are conflicting results in the field with respect to the question whether RanBP2 phosphorylation is a requirement for the interaction with BicD2 (Baffet et al, 2015;Gibson et al, 2021). Our results now reconcile these reports, because they show that BicD2 can bind both non-phosphorylated as well as phosphorylated RanBP2DDB.…”

“…RanBP2 (or Nup358) is known to recruit BicD2 through a small disordered region between two Ran binding domains as demonstrated in yeast two-hybrid assays (Splinter et al, 2010). The BicD2 binding domain of RanBP2 (hereafter RanBP2BBD) interacts with the C-terminus of BicD2 (715-804) (Gibson et al, 2021) and contains several consensus CDK1 phosphorylation sites, which have been shown to be important for BicD2 binding (Baffet et al, 2015). The individual contribution of these phosphorylation sites to BicD2 binding is unknown.…”

“…The individual contribution of these phosphorylation sites to BicD2 binding is unknown. A recent in-vitro experiment demonstrated that the RanBP2BBD-BicD2 complex can also be formed in the absence of phosphorylation (Gibson et al, 2021). Since all these experiments used the truncated BicD2 C-terminus, the binding of the full-length BicD2 to RanBP2BBD remains to be characterized.…”

In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2

“…RanBP2 (or Nup358) is known to recruit BicD2 through a small disordered region between two Ran binding domains (2003-2444) as demonstrated in yeast two-hybrid assays. 7 The BicD2 binding domain of RanBP2 (hereafter RanBP2BBD) interacts with the C-terminus of BicD2 (715-804) 36 and contains several consensus CDK1 phosphorylation sites, which have been shown to be important for BicD2 binding. 9 However, a recent in-vitro experiment demonstrated that the RanBP2 BBD -BicD2 complex can also be formed in the absence of phosphorylation.…”

“…9 However, a recent in-vitro experiment demonstrated that the RanBP2 BBD -BicD2 complex can also be formed in the absence of phosphorylation. 36 Since all these experiments used the truncated BicD2 Cterminus, the binding of the full-length BicD2 to RanBP2 BBD remains to be characterized. In order to clarify the role of phosphorylation in BicD2-RanBP2 BBD complex formation, and to confirm the biochemical activity of our recombinant full-length BicD2, we evaluated the interaction with its binding partner RanBP2.…”