Paclitaxel

Lyophilized tubulin (Cytoskeleton, Inc.) was solubilised in Brb80 buffer (80 mM PIPES, 2 mM MgCl2, 1 mM EGTA, pH 6.8, 1 mM NaN3, 1 mM DTT, pH 6.8), to a final concentration of 2 mg/mL. The polymerization was induced by adding 1 mM Guanosine-5′-triphosphate (GTP) and incubation for 15 min at 30 °C. Then, 20 μM paclitaxel (taxol, SIGMA) was used to stabilize the MT and incubation took place for another 15 min at 30 °C. The MT were spun down at 180.000 × g (Beckman TLA-55 rotor) for 30 min at 30 °C and the pellet was resuspended in warm Brb80 buffer with 20 μM paclitaxel. Subsequently, a 1:1 ratio of ¹³C¹⁵N tau K32 was added. The interaction partners were incubated for 30 min at 37 °C. In the following isotopically labelled tau K32 in complex with MT was separated from the unbound, non-polymerised fraction by centrifugation at 180.000 xg (Beckman TLA-55 rotor) for 30 min at 30 °C. Afterwards, the pellet was washed with 40 mM phosphate buffer, pH 7, with protease inhibitor (as described earlier) and 1 mM NaN3, without disturbing the pellet. A 1.3 mm rotor was packed with the pellet.
Basic experimental details for each sample used in this manuscript. Further details are provided in the materials and methods section, and Table S1.

A polymerization mixture was prepared with BRB80 + 32 μM tubulin + 1 mM GTP + 4 mM MgCl₂ + 5% DMSO. The mixture was incubated on ice for 5 min, followed by incubation at 37°C for 30 min. The polymerized microtubules were diluted into prewarmed BRB80 + 10 μM paclitaxel, centrifuged at 110,000 rpm (199,000 × g) in an Airfuge (Beckman-Coulter), and resuspended in BRB80 + 10 μM paclitaxel (99.5+%, GoldBio).

For the preparation of solid-state NMR samples lyophilized tubulin (Cytoskeleton, Inc.) was solubilised in BRB80 buffer (80 mM PIPES, 2 mM MgCl₂, 1 mM EGTA, pH 6.8, 1 mM NaN₃, 1 mM DTT, pH 6.8 supplemented with protease inhibitor (Sigma-Aldrich, cOmplete EDTA-free), to a final concentration of 2 mg/mL). Then 1 mM Guanosine-5’-triphosphate (GTP) was added and incubation took place for 15 min at 30 °C. In the following, 20 μM paclitaxel (Taxol, SIGMA) was used to stabilize the MT and incubation took place for another 15 min at 30 °C. The MT were spun down at 180,000 g (Beckman TLA-55 rotor) for 30 min at 30 °C and the pellet was resuspended in warm BRB80 buffer with 20 μM paclitaxel. Subsequently 0.55 mg/mL [¹³C-¹⁵N]-MAP7 MTBD was added. The interaction partners were incubated for 30 min at 30 °C. In the following step [¹³C-¹⁵N]-MAP7 MTBD in complex with MT was separated from the unbound, non-polymerised fraction by centrifugation at 180.000 g (Beckman TLA-55 rotor) for 30 min at 30 °C. Afterwards, the pellet was washed with BRB80 buffer containing protease inhibitor, without disturbing the pellet. A 1.3 mm rotor was packed with the pellet.