title: Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies.
abstract: Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains. The molecular mechanism of action of doublecortin is only incompletely understood. Anti-doublecortin antibodies, such as the rabbit polyclonal Abcam 18732, are widely used as neurogenesis markers. Here, we report the generation and characterization of antibodies that bind to single doublecortin domains. The antibodies were used as tools to obtain structures of both domains. Four independent crystal structures of the N-terminal domain reveal several distinct open and closed conformations of the peptide linking N- and C-terminal domains, which can be related to doublecortin function. An NMR assignment and a crystal structure in complex with a camelid antibody fragment show that the doublecortin C-terminal domain adopts the same well defined ubiquitin-like fold as the N-terminal domain, despite its reported aggregation and molten globule-like properties. The antibodies' unique domain specificity also renders them ideal research tools to better understand the role of individual domains in doublecortin function. A single chain camelid antibody fragment specific for the C-terminal doublecortin domain affected microtubule binding, whereas a monoclonal mouse antibody specific for the N-terminal domain did not. Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryo-electron micrographs is the C-terminal domain rather than the N-terminal one.
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Dominique
authors.initials: D
authors.lastname: Burger
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Martine
authors.initials: M
authors.lastname: Stihle
authors.affiliation: the Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland.
authors.forename: Ashwani
authors.initials: A
authors.lastname: Sharma
authors.affiliation: pRED Pharma Research and Early Development, Small Molecule Research, Discovery Technologies, Roche, Nutley, New Jersey 07110.
authors.forename: Paola
authors.initials: P
authors.lastname: Di Lello
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Jörg
authors.initials: J
authors.lastname: Benz
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Brigitte
authors.initials: B
authors.lastname: D'Arcy
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium, and the Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Maja
authors.initials: M
authors.lastname: Debulpaep
authors.affiliation: pRED Pharma Research and Early Development, Small Molecule Research, Discovery Technologies, Roche, Nutley, New Jersey 07110.
authors.forename: David
authors.initials: D
authors.lastname: Fry
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Walter
authors.initials: W
authors.lastname: Huber
authors.affiliation: Roche Pharmaceutical Research and Early Development, NORD Discovery and Translational Area, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kremer
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium, and the Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Toon
authors.initials: T
authors.lastname: Laeremans
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Hugues
authors.initials: H
authors.lastname: Matile
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Alfred
authors.initials: A
authors.lastname: Ross
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Arne C
authors.initials: AC
authors.lastname: Rufer
authors.affiliation: Roche Pharmaceutical Research and Early Development, NORD Discovery and Translational Area, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Schoch
authors.affiliation: the Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland.
authors.forename: Michel O
authors.initials: MO
authors.lastname: Steinmetz
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium, and the Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Markus G
authors.initials: MG
authors.lastname: Rudolph
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and.
authors.forename: Ralf
authors.initials: R
authors.lastname: Thoma
authors.affiliation: From the pRED Pharma Research and Early Development, Therapeutic Modalities, and armin.ruf@roche.com.
authors.forename: Armin
authors.initials: A
authors.lastname: Ruf
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D002162:Camelus; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D000089622:Doublecortin Domain Proteins; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D009479:Neuropeptides / Q000737:chemistry*; D000072417:Protein Domains; D020836:Protein Structure, Quaternary; D011817:Rabbits; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000089622:Doublecortin Domain Proteins; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; D057127:Single-Chain Antibodies
keywords: cell migration; crystal structure; microtubule; microtubule-associated protein (MAP); neurogenesis; neuroscience; protein conformation; protein stability; protein-protein interaction; structural biology