Structural Studies of the Doublecortin Family of MAPs - Methods in Cell Biology - page 41

UCSF Chimera ( Pettersen et al., 2004 ) was used for visualization of 3D models
and rigid-body fitting of atomic structures in the cryo-EM volumes. Several crystal
structures of a - and b -tubulin (1JFF.pdb ( L¨we, Li, Downing, & Nogales, 2001 ),
3HKE.pdb ( Dorl´ans et al., 2009 )) were fitted independently in their respective
densities in the DCX-K-MT reconstruction because we wanted to evaluate the
conformation of each monomer separately in our DCX-stabilized MTs formed in
the absence of paclitaxel. The quality of the fits was assessed by calculating the
cross-correlation between our structure and a simulated 8 ˚ model for each atomic
structure: b -tubulin from 1JFF gave the best score of 0.651 in our b -tubulin density
(3HKE b -tubulin chain B gave 0.599), a -tubulin from 1JFF and 3HKE chain A gave
similar scores, respectively, 0.605 and 0.607, whereas a chimeric structure increased
the cross-correlation slightly to 0.614 (3HKE chain A residues 31-61, 69-92, and
275-298, corresponding to the N, H2S3, and M loop regions, respectively, involved
in lateral contact formation) were substituted into 1JFF chain A. To build the pseudo-
atomic model of the DCX-MT complex, four tubulin subunits and the N-DC domain
of DCX (1MJD.pdb, model 11, residues 46-140) were placed independently in
the EM density using Chimera, and Flex-EM was used for the refinement of the
multiple subunit fitting ( Topf et al., 2008 ). The best multicomponent fit had a
cross-correlation value of 0.819 (PDB ID 2XRP). This pseudo-atomic model allowed
us to visualize the precision with which the DC domain contacts four tubulin
dimers at its binding site and revealed several disease-causing mutations in both
DCX and tubulin at their binding interface ( Bahi-Buisson et al., 2013; Fourniol
et al., 2010 ).
3.2.3.8 Insight into MT structure in the absence of stabilizing drugs
High-resolution information about MTs and straight pf structures has mainly been
derived from paclitaxel-stabilized tubulin assemblies ( Li, DeRosier, Nicholson,
Nogales, & Downing, 2002; L¨we et al., 2001 ). In fact, paclitaxel appears to be a
promiscuous stabilizer, binding MTs with varying pf numbers, sheets of antiparallel
and inverted pfs ( Nogales, Wolf, & Downing, 1998 ), or short-straight pfs in solution
( Elie-Caille et al., 2007 ). These data imply that paclitaxel stabilizes a conformation
of tubulin that favors straight pfs but that is not necessarily specific to MTs. How-
ever, because there has been no high-resolution model of MTs in the absence of
paclitaxel, it has been unclear whether the conformation stabilized by the drug
is a native state of tubulin or is induced by drug binding. We found that the
paclitaxel-binding pocket is present in our reconstructions, demonstrating that
paclitaxel stabilizes a native conformation of polymerized tubulin ( Fig. 3.5 C).
Our data thus reveal a native structure of MTs bound solely by cellular ligands, where
the straight pf structure appears virtually identical to that found in zinc-induced
sheets.
Our structure confirms the importance of the lateral contacts formed by the M, N,
and H2S3 loops previously visualized by Li et al. (2002) and more recently by Sui
and Downing (2010) . While the procedure employed by Downing and colleagues in
these studies averaged together a - and b -tubulin, our reconstruction discriminates