title: Structural studies of the doublecortin family of MAPs.
abstract: Doublecortin (DCX) is a microtubule (MT)-stabilizing protein essential for neuronal migration during human brain development. Missense mutations in DCX cause severe brain defects. This implies that the many other MT-stabilizing proteins in neurons cannot compensate for DCX function. To understand the unusual properties of DCX, we expressed the recombinant human DCX in Sf9 cells and undertook structural characterization of its interaction with MTs using cryo-electron microscopy. DCX specifically nucleates 13-protofilament (13-pf) MTs, the architecture of human MTs in vivo. Cryo-electron tomography (cryo-ET) of DCX-nucleated MTs showed that they are primarily built from B-lattice contacts interrupted by a single discontinuity, the seam. Because of this asymmetry, we used single-particle reconstruction and determined the 8Å structure of DCX-stabilized 13-pf MTs in the absence of a stabilizing drug. The DCX-binding site, at the corner of four tubulin dimers, is ideally suited to stabilize both lateral and longitudinal tubulin lattice contacts. Its precise geometry suggests that DCX is sensitive to the angle between pfs, and thereby provides insight into the specificity of DCX for 13-pf MT architecture. DCX's precise interaction at the corner of four tubulin dimers also means that DCX does not bind the MT seam. Our work has provided mechanistic insight into the evolutionarily conserved DCX family of MT-stabilizing proteins and also into more general regulatory mechanisms of the MT cytoskeleton.
authors: Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom.
authors: Franck
authors: F
authors: Fourniol
authors: Mylène
authors: M
authors: Perderiset
authors: Anne
authors: A
authors: Houdusse
authors: Carolyn
authors: C
authors: Moores
mesh_terms: D001665:Binding Sites; D001921:Brain / Q000196:embryology / Q000378:metabolism*; D002465:Cell Movement / Q000235:genetics; D054221:Classical Lissencephalies and Subcortical Band Heterotopias / Q000235:genetics; D020285:Cryoelectron Microscopy / Q000379:methods; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D000089742:Doublecortin-Like Kinases; D055032:Electron Microscope Tomography / Q000379:methods; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D008870:Microtubules / Q000378:metabolism*; D008958:Models, Molecular; D009474:Neurons / Q000378:metabolism; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism
chemical_list: C000714328:DCX protein, human; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; C117347:DCLK1 protein, human; D000089742:Doublecortin-Like Kinases; C501017:DCLK2 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: Cryo-electron microscopy; Doublecortin; Image reconstruction; Lissencephaly; Microtubules; Neuronal MAPs