Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Abstract

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathminlike domain of the RB3 protein (T 2 RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulinstathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T 2 RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T 2 RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T 2 RB3, T 2 Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.Microtubules are essential for eukaryotic chromosome segregation, cellular architecture and intracellular trafficking, among other processes. Understanding microtubule dynamics, regulation, and organization requires knowledge of the nucleotide-regulated assembly switch of tubulin. Microtubules are hollow cylinders made of protofilaments of ␣␤-tubulin dimers in head to tail association, forming a pseudohelical lattice (1). The functional assembly-disassembly cycle of a ␣␤-tubulin molecule includes activation by GTP binding at the ␤-subunit, polymerization into microtubules, GTP hydrolysis at the ␤-␣ interdimer interface and depolymerization of GDP-tubulin, followed by replacement by GTP. Vectorial polymerization and GTP hydrolysis combine with tubulin structural plasticity in microtubule dynamics (2). Depolymerizing microtubule ends show characteristic curled protofilaments, whereas relatively straight sheets form at growing ends (3, 4). GDP-tubulin does not assemble into microtubules, but forms double rings (5), which also form upon microtubule depolymerization (6) and correspond to curved microtubule protofilaments (7,8). The tendency of GDP-tubulin to curve is thought to strain the microtubule lattice, causing disassembly when the terminal cap of GTP-bound tubulin...

Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin

“…Taken together, these observations suggest that stathmin can directly promote catastrophe by binding to and acting upon the exposed protofilaments that are predicted to exist at the tips of growing MTs (9); consideration of the existing stathmin-tubulin complex structures (23)(24)(25)(26) suggests that it acts by inhibiting lateral interactions between the PFs. It is also possible that stathmin increases the GTPase of the tubulin subunits in the protofilaments to which it is bound (27).…”

“…Such an inhibition could occur because stathmin's binding site is close to the interface between protofilaments (23-25). Alternatively, because both DL-rings and the T 2 S complex are curved (15)(16)(17)(18)26), stathmin could inhibit lateral interactions by inducing in protofilaments a degree of curvature that is incompatible with their incorporation into the lattice.…”

Tau Binds to Multiple Tubulin Dimers with Helical Structure

“…RB3-tubulin includes a longitudinal interaction between tubulin dimers. 18 The similar size of the tau-RB3-tubulin and tau-tubulin complexes indicates that tau is able to mediate these longitudinal tubulin interactions as well (Figure 4). This is in agreement with a recent NMR study which demonstrated longitudinal assembly of tubulin dimers by a tau fragment.…”

Design and Characterization of Modular Scaffolds for Tubulin Assembly

“…Proteins were overexpressed in Escherichia coli BL21 DE3 Star, in LB medium supplemented with kanamycin, using 0.5 mM isopropyl ␤-D-1-thiogalactopyranoside to induce an expression period of 3 h at 37°C. Purification was as described (6) except that a first step of nucleic acid precipitation by spermine (24) was added and that the heating step was omitted for R4 and R4a. The concentration of purified SLD was determined by measuring the absorbance at 280 nm, taking advantage of the presence of tryptophan residues in these constructs, as opposed to wild type RB3 SLD , whose absorbance at 280 nm is very weak.…”