Mechanism of Microtubule Stabilization by Doublecortin Carolyn A Moores, Mylène Perderiset, Fiona Francis, Jamel Chelly, Anne Houdusse, Ronald A Milligan  Molecular Cell  Volume 14, Issue 6, Pages 833-839 (June 2004) DOI: 10.1016/j.molcel.2004.06.009

Figure 1 Doublecortin Selectively Binds 13 Protofilament Microtubules and Nucleates Microtubules (A) Domain organization of doublecortin. N-DC and C-DC each comprise 85 residues. The C-terminal Ser/Pro-rich domain is made up of 100 residues, the majority of which is missing in the t-DCX construct used to determine the doublecortin binding site. (B) t-DCX (gray bars) and DCX (white bars) show binding selectivity for specific MT architectures. Plotted values are percent total MTs of a given pf number that showed an 80 Å−1 signal in the optical diffractometer. Total number of MTs examined was 361 and 224 for t-DCX and DCX decoration, respectively. (C) MTs copolymerized with doublecortin have predominantly 13 pfs (white bars). Polymerization of pure tubulin results in mostly 14 pf MTs (black bars) (total polymer length, 354 μm and 353 μm, respectively). For cryo-electron microscopy, 25 μM tubulin was copolymerized with 34 μM full-length DCX at 37°C for 6 hr prior to freezing. (D) Typical negative stain image of the result of tubulin polymerization in the absence (top panel) and presence (bottom panel) of DCX. Scale bar, 1 μm. (E) Average length of MTs polymerized in the absence (−DCX) and presence (+DCX) of full-length doublecortin, compared to MTs polymerized with GMPCPP (measured from negative stain EM images). Molecular Cell 2004 14, 833-839DOI: (10.1016/j.molcel.2004.06.009)

Figure 2 Doublecortin Binds in the Valley between Microtubule Protofilaments (A) Front view of the 3D difference map (yellow) representing t-DCX, displayed with a 3D map of the undecorated MT (blue). The difference map was obtained by subtracting the 3D map of the undecorated MT from a 3D map of t-DCX-decorated MT. Doublecortin binds between tubulin pfs and lies over one of the two distinct fenestrations in the MT wall. (B) View from the plus end showing how doublecortin fits snugly into the valley between adjacent pfs. Orange and green areas approximately delineate the kinesin and MAP2/tau binding sites respectively. The asterisk shows the location of the taxol binding site. (C) Close-up view of (A), with the atomic model of two tubulin heterodimers (Löwe et al., 2001) docked into the MT map. The yellow density attributable to doublecortin lies at the junction of four tubulin monomers (labeled α1, β1, α2, and β2). We emphasize that our data do not allow us to tell whether doublecortin binds at the location shown—between two heterodimers or at the similar but distinct site 40 Å away—at the junction between four heterodimers (asterisk). (D) View of (C) from the MT plus end shows that doublecortin makes relatively sparse contacts with individual monomers, suggesting that its binding site is unique to polymerized tubulin. (E) The doublecortin difference map can only accommodate a single DC domain (left). The right panel shows N-DC in an equivalent orientation and illustrates that surface residues mutated in lissencephaly (R78, D86, R89, R102, shown in space-filling representation) map to one side of the DC domain and are likely involved in MT binding. (Scale bars, 20 Å). Molecular Cell 2004 14, 833-839DOI: (10.1016/j.molcel.2004.06.009)