Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies

Burger, Dominique; Stihle, M.; Sharma, Ashwani; Lello, Paola Di; Benz, Jörg; D’Arcy, Brigitte; Debulpaep, Maja; Fry, David C.; Huber, Walter; Kremer, Thomas; Laeremans, Toon; Matile, Hugues; Ross, Alfred; Rufer, Arne C.; Schoch, Guillaume A.; Steinmetz, Michel O.; Steyaert, Jan; Rudolph, M.G.; Thoma, Ralf; Ruf, A.

doi:10.1074/jbc.m116.726547

Cited by 17 publications

(52 citation statements)

References 41 publications

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“…4A, cyan, root-mean-square deviation, RMSD = 1.3 Å). In contrast, the conformation of the MT-bound CDC in our reconstruction is substantially different from that stabilized in its crystal structure (Burger et al, 2016) (Fig. 4A, yellow, RMSD = 4.3 Å).…”

Section: -Pfcontrasting

confidence: 70%

“…11), and therefore not suitable for the high-resolution structure determination (Manka and Moores, 2018). Docking of CDC in the conformation found in the crystal structure (Burger et al, 2016) to a minimal (4-dimer) bent tubulin assembly ( Supplementary Fig. 10A-C) and the docking refinement by Rosetta illuminates the probability of this hypothesised interaction (Fig.…”

Section: -Pfmentioning

confidence: 99%

“…12). To build the assembly, we used the only available crystal structure of a bent tubulin in the GTP state (PDB: 3RYF) (Nawrotek et al, 2011) and the only available crystal structure of intact CDC (PDB: 5IP4) (Burger et al, 2016), as explained in Supplementary Fig. 10.…”

Section: -Pfmentioning

confidence: 99%

“…An obvious difference between the two DC domains is the length of loop 3, which is 3 residues shorter in CDC ( Fig. 1B), while structural studies of individual domains suggest that the biological properties of NDC and CDC are distinct (Burger et al, 2016;Cierpicki et al, 2006;Kim et al, 2003;Rufer et al, 2018). Isolated DC domains alone do not stimulate MT polymerisation, i.e.…”

Section: Introductionmentioning

confidence: 97%

“…Isolated DC domains alone do not stimulate MT polymerisation, i.e. a DC domain tandem is required for this activity (Taylor et al, 2000), and a distinct contribution of each DC domain to MT lattice-based function of DCX has been indicated (Bechstedt and Brouhard, 2012;Bechstedt et al, 2014;Burger et al, 2016;Liu et al, 2012), although the mechanism by which this occurs is also not known.…”

Section: Introductionmentioning

confidence: 99%

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

Manka

Moores

2019

Preprint

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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)

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Section: -Pfcontrasting

confidence: 70%

Section: -Pfmentioning

confidence: 99%