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

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

doi:10.1074/jbc.m116.726547

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.

Highlights

Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryoelectron micrographs is the C-terminal domain rather than the N-terminal one
A conformational switching model between an open and a closed conformation of doublecortin was previously proposed with the sequence region 221–245 that connects the DCX domains expanding or folding back on the N-DCX core and thereby modulating the distance between the N-DCX and C-DCX domains [13]
This model postulates that the resulting changes in the distance between N-DCX and C-DCX are of functional importance in the control of tubulin polymerization and microtubule bundling

Summary

Introduction

This suggests that the microtubule-interacting doublecortin domain observed in cryoelectron micrographs is the C-terminal domain rather than the N-terminal one. From two clusters of mutations in X-linked lissencephaly and subcortical band heterotopia patients, an internal tandem repeat of domains with 27% sequence identity was identified on the doublecortin gene, DCX. These domains were detected in sequences of other proteins and are called DCX domains. Similar experiments showed that single DCX molecules recognize the ends of growing microtubules by their concave lattice curvature and that only patient mutations on C-DCX abolish this [12] These mutations on C-DCX map to the contact sites between the DCX domain and microtubules observed in the cryo-EM reconstructions, suggesting that the bound DCX domain could be C-DCX instead of N-DCX [8, 12]. The C-terminal domain of doublecortin domain-containing protein 2 (DCDC2) has 32% sequence identity with C-DCX and has a DCX fold according to its NMR structure with the PDB code 2DNF

Objectives

Results

Conclusion