Pseudo-repeats in doublecortin make distinct mechanistic contributions to microtubule regulation

Doublecortin (DCX) is a neuronal microtubule-associated protein (MAP) indispensable for brain development. Its flexibly linked DC domains – NDC and CDC – mediate microtubule (MT) nucleation and stabilisation, but it is unclear how. Using high-resolution time-resolved cryo-EM, we mapped NDC and CDC interactions with tubulin at different MT polymerisation stages and studied their functional effects on MT dynamics using TIRF microscopy. Although coupled, each DC repeat appears to have a distinct role in MT nucleation and stabilisation by DCX: CDC is a conformationally plastic tubulin binding module that appears to facilitate MT nucleation by binding tubulin oligomers and stabilising tubulin-tubulin contacts in the nascent MT lattice, while NDC appears to be favoured along the mature lattice, providing enhanced and durable MT stabilisation. Our near-atomic resolution structures of MT-bound DC domains also explain in unprecedented detail the DCX mutation-related brain defects observed in the clinic. This modular composition of DCX reflects a common design principle among MAPs where pseudo-repeats of tubulin/MT binding elements chaperone or stabilise distinct conformational transitions to regulate distinct stages of MT dynamic instability.