Structural Studies of the Doublecortin Family of MAPs - Methods in Cell Biology - page 29

blindness) and whose MT-stabilizing activity is essential for photoreceptor cell de-
velopment ( Liu, Zuo, & Pierce, 2004 ). In addition, Dcdc2 , another DCX-MAP
encoding gene, is linked with developmental dyslexia ( Meng et al., 2005 ), and its
protein product is involved with the control of primary cilia size and activity in neu-
rons ( Massinen et al., 2011 ).
The DCX 366-amino-acid sequence (40 kDa) shows no homology to other
classical neuronal MAPs. In fact, sequence analysis reveals that DCX is built from
an N-terminal tandem of homologous 90-amino-acid (11 kDa) domains which were
accordingly named DC domains; these domains are separated by a well-conserved
but presumed unstructured linker and are followed by a presumed intrinsically un-
structured C-terminal serine/proline-rich (S/P-rich) domain ( Fig. 3.1 ). Point muta-
tions causing lissencephaly cluster within the two DC domains and modify the
DCX-MT interaction in transfected cells ( Bahi-Buisson et al., 2013; Sapir et al.,
2000; Taylor, Holzer, Bazan, Walsh, & Gleeson, 2000 ). These data established
the DC domains of DCX as MT-binding domains and reinforced the importance
of DCX-MT binding during neuronal cortical migration.
An NMR study revealed the solution, b -grasp-like structure of the recombinant
N-terminal DC domain (N-DC), and initiated the exploration of structure-function
relationships of disease-causing point mutations in this domain ( Kim et al., 2003 ). This
analysis allowed discrimination between mutations of buried residues—affecting
folding and stability of the protein—and of surface residues affecting putative inter-
actions with DCX's binding partners, including tubulin and MTs. However, an equiv-
alent study of C-DC and of longer constructs proved technically challenging, leaving
many aspects of DCX's unique MT-binding mechanism unresolved.
3.1 RATIONALE
We were fascinated by the idea that although neurons are full of MT-stabilizing
proteins, DCX has unique properties that cannot be functionally compensated
for by other neuronal MAPs. To address this mystery, we used established structural
approaches for studying MAPs ( Amos & Hirose, 2007; Hoenger & Gross, 2008 ).
Our early work revealed several distinctive properties of DCX and its interaction
with MTs ( Moores et al., 2004, 2006 ). An emerging area of interest is the sensitivity
of MAPs to tubulin-bound nucleotide ( Maurer, Bieling, Cope, Hoenger, & Surrey,
2011; Zanic, Stear, Hyman, & Howard, 2009 ); however, we found that DCX stabi-
lizes MTs independent of the bound nucleotide, it does not affect the intrinsic
GTPase of tubulin polymer, and the lattice parameters of the DCX-stabilized MTs
reflect the nucleotide that is bound in the lattice ( Fourniol et al., 2010 ). In our hands,
DCX does not enhance MT growth rates but blocks depolymerization ( Moores et al.,
2006 ). All of these effects occur at substoichiometric or close to stoichiometric ratios
of DCX:tubulin and appear to operate independent of MT bundling, which is the
primary and apparently nonspecific outcome of DCX-MT interactions at superstoi-
chiometric ratios of DCX both in vivo and in vitro ( Sapir et al., 2000 ).