Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C-terminal domain

Abstract

Doublecortin (DCX) is a microtubule (MT) associated protein that regulates MT structure and function during neuronal development and mutations in DCX lead to a spectrum of neurological disorders. The structural properties of MT-bound DCX that explain these disorders are incompletely determined. Here, we describe the molecular architecture of the DCX-MT complex through an integrative modeling approach that combines data from X-ray crystallography, cryo-EM and a high-fidelity chemical crosslinking method. We demonstrate that DCX interacts with MTs through its N-terminal domain and induces a lattice-dependent self-association involving the C-terminal structured domain and its disordered tail, in a conformation that favors an open, domain-swapped state. The networked state can accommodate multiple different attachment points on the MT lattice, all of which orient the C-terminal tails away from the lattice. As numerous disease mutations cluster in the C-terminus, and regulatory phosphorylations cluster in its-tail, our study shows that lattice-driven self-assembly is an important property of DCX.

Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking

“…In addition, our MT-binding assay demonstrates that, while C-DCX itself does not bind MTs, C-DCX increases the interactions of DCX with MTs ( Figure 3B ). This suggests that the interactions between DCX and MTs are enhanced by DCX’s C-terminal domain, which is consistent with recent findings that the tail of DCX (amino acid 303 to the C-terminal end) helps to maintain the associations between DCX molecules on the MT lattice ( Rafiei et al, 2022 ).…”

“…Therefore, the overall conformation of DCX changes when DCX associates with MTs. Studies also showed that DCX dynamically associates with MTs mainly through its N-terminal R1 MT-binding domain ( Moslehi et al, 2017 ; Rafiei et al, 2022 ). However, DCX’s C-terminal region still plays a critical role in the association of DCX with MTs despite C-DCX’s inability to directly bind to MTs ( Rafiei et al, 2022 ).…”

“…DCX binds MTs in a dimerized form or as oligomers of higher order, and it has been shown that this DCX self-association is an MT-dependent phenomenon ( Moores et al, 2006 ; Rafiei et al, 2022 ). Therefore, the overall conformation of DCX changes when DCX associates with MTs.…”

“…Studies also showed that DCX dynamically associates with MTs mainly through its N-terminal R1 MT-binding domain ( Moslehi et al, 2017 ; Rafiei et al, 2022 ). However, DCX’s C-terminal region still plays a critical role in the association of DCX with MTs despite C-DCX’s inability to directly bind to MTs ( Rafiei et al, 2022 ). The C-terminus of DCX facilitates the binding of neighboring DCX molecules to MTs through its interactions with the N-terminal domain of the neighboring DCX ( Rafiei et al, 2022 ).…”

“…However, DCX’s C-terminal region still plays a critical role in the association of DCX with MTs despite C-DCX’s inability to directly bind to MTs ( Rafiei et al, 2022 ). The C-terminus of DCX facilitates the binding of neighboring DCX molecules to MTs through its interactions with the N-terminal domain of the neighboring DCX ( Rafiei et al, 2022 ). This suggests that the C- and N-terminal domains of DCX have an intrinsic affinity for each other (possibly resulting in a ‘closed’ conformation as discussed above) and that MT binding leads to a shift from intramolecular interactions between the N-terminal and C-terminal domains to intermolecular interactions between the domains of neighboring MT-bound DCX molecules.…”