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 remain poorly resolved. 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 both the C-terminal structured domain and the C-tails, 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-tails away from the lattice. As numerous disease mutations cluster in the C-terminus, and regulatory phosphorylations cluster in the C-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

“…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).…”

“…Collectively, our in vitro reconstitution studies with purified proteins agree with our in vivo observations that DCX downregulates dynein’s activity, and that the C-terminus of DCX auto-inhibits DCX’s interaction with dynein. Indeed, a recent study has shown that the C-terminus of DCX facilitates the binding of neighboring DCX molecules to MTs via intermolecular interactions with DCX’s N-terminal domain (Rafiei et al, 2022), which suggests that the C- and N-terminal domains of DCX have an intrinsic affinity for each other.…”

High sensitivity proteome-scale search for crosslinked peptides using CRIMP 2.0

“…For example, structure determination requires extensive crosslinking to produce enough constraints for accurate model building. Such an exercise has a moderate tolerance for false positives, provided that the crosslinks sample the structure in an unbiased manner 15,16 . On the other hand, defining an in situ protein network could be achieved with as little as one crosslink per interaction.…”