Microtubules (MTs) play a fundamental role in many vital processes such as cell division and neuronal activity. They are key structural and functional elements in axons, supporting neurite differentiation and growth, as well as transporting motor proteins along the axons, which use MTs as support tracks. Tau is a stabilizing MT associated protein, whose functions are mainly regulated by phosphorylation. A disruption of the MT network, which might be caused by Tau loss of function, is observed in a group of related diseases called tauopathies, which includes Alzheimer’s disease (AD). Tau is found hyperphosphorylated in AD, which might account for its loss of MT stabilizing capacity. Since destabilization of MTs after dissociation of Tau could contribute to toxicity in neurodegenerative diseases, a molecular understanding of this interaction and its regulation is essential.

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...