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

Abstract

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 pseudorepeats of tubulin/MT binding elements chaperone or stabilise distinct conformational transitions to regulate distinct stages of MT dynamic instability.Keywords: cryo-EM / doublecortin / DCX domain / microtubule-associated protein / pseudorepeat NDC binding to MT lattice is not selective for a particular MT architectureSince NN-stimulated MT nucleation produces 14-PF almost as well as 13-PF MTs, we investigated whether there were any architecture-specific differences in NDC observed in these MTs. The manual collection of NN-MT cryo-EM data for high-resolution 3D reconstruction was focused on the physiological 13-PF architecture to enable direct comparisons with WT-MT reconstruction. Nevertheless, supervised 3D classification of all picked NN-MT segments revealed a 13-PF class containing 73 % and a 14-PF class containing 26 % of segments. The remaining 1 % are particles of relatively poor quality. The 14-PF NN-MT reconstruction had a resolution nearly equivalent (3.9 Å)

Autoregulatory control of microtubule binding in the oncogene, doublecortin-like kinase 1

“…Within the microtubule-binding region of DCLK1 (aa 44-263), we found that 9 sites were more frequently phosphorylated in DCLK1-WT samples and 17 sites were more frequently phosphorylated in DCLK1-ΔC samples ( Figure 3A-B). Of the 17 phosphorylation sites in DCLK1-ΔC, 5 either directly contact tubulin or are adjacent to residues that directly contact tubulin within the lattice (42). The architecture of DCLK1 suggests it likely has the flexibility to autophosphorylate its N-terminal half due to an intrinsically disordered region between the DC2 domain and the kinase domain ( Figure 3C, aa 263-374).…”

“…Although originally hypothesized to be DC1, subsequent structural and biochemical studies called into question the identity of the bound DC domain (44,45). A recent cryo-EM study has proposed that DC2 binds to the newly polymerized GTP microtubule lattice, while DC1 binds the mature GDP microtubule lattice (42). All of our binding assays are performed with taxol-stabilized microtubules in the GDP state (46); however, based on our phosphorylation data, we reasoned that DC2 could still contribute to efficient binding of DCLK1 to GDP microtubules.…”

“…All of our binding assays are performed with taxol-stabilized microtubules in the GDP state (46); however, based on our phosphorylation data, we reasoned that DC2 could still contribute to efficient binding of DCLK1 to GDP microtubules. In order to test if phosphorylation of the DC2 domain is responsible for the dramatic decrease in the microtubule-binding affinity of DCLK1-ΔC, we mutated 4 conserved residues within the DC2 domain DCLK1-ΔC 4A ) that showed an increase in phosphorylation and also sit at or adjacent to the tubulin-binding interface ( Figure 4A, S3A) (42). We reasoned that if phosphorylation of these residues is responsible for the decreased microtubule-binding affinity of DCLK1-ΔC, then mutating these residues to alanines, which cannot be phosphorylated, should rescue the microtubule-binding defect of this construct in the presence of ATP.…”

“…However, a subsequent study showed that specifically blocking DC2, but not DC1, prevented DCX from interacting with microtubules, suggesting DC2 is critical for lattice binding (44). Recent cryo-EM data of DC1 and DC2 bound to microtubule lattices in different nucleotide states unveiled that DC2 binds to the GTP microtubule lattice, while DC1 prefers the GDP microtubule lattice (42). This model is consistent with observations that DCX tracks the growing plusend of the microtubule, which is specifically disrupted by missense mutations in DC2 (33).…”

A doublecortin-domain protein of Toxoplasma and its orthologues bind to and modify the structure and organization of tubulin polymers

“…Interaction with microtubules differs dramatically between TgDCX and its orthologues, also likely differs between TgDCX and human doublecortin. In the case of doublecortin, a detailed model of the interaction of its two DCX domains (NDC and CDC) with microtubules has been proposed, based on high-resolution structural information from cryoEM [32]. A striking feature of the model is that for the most part, binding of the two domains to the microtubule is mutually exclusive: either NDC binds or CDC binds, with differing consequences for microtubule architecture, but apparently the two domains rarely or never bind simultaneously.…”