Structure of tubulin at 6.5 Å and location of the taxol-binding site

TUBULIN, the major component of microtubules, is a heterodimer of two chains, α and β¹, both of relative molecular mass 50,000 (M_r50K) and with 40-50% identity. The isotypic variety² and conformational flexibility of tubulin have so far made it impossible to obtain crystals for X-ray work³. Structural knowledge of tubulin has been limited to about 20 Å from X-ray diffraction of oriented microtubules⁴, and from electron microscopy of microtubules and zinc-induced crystalline sheets in negative stain^5,6. The sheets consist of protofilaments similar to those in microtubules but associated in an antiparallel arrangement⁷, and their two-dimensional character is ideal for high-resolution electron microscopy^8,9. Here we present a three-dimensional reconstruction of tubulin to 6.5 Å resolution, obtained by electron crystallography of zinc-induced two-dimensional crystals of the protein. The α- and β-subunits appear topologically similar, in agreement with their sequence homology¹⁰. Several features can be defined in terms of secondary structure. An apparent α-helical portion, adjacent to both interdimer and inter-protofilament contacts, is tentatively attributed to a segment near the carboxy terminus of the protein. We can assign the α- and β-subunits on the basis of projection studies of the binding of taxol*, which show one taxol site per tubulin heterodimer, in agreement with the known stoichiometry of taxol in microtubules¹¹. These studies indicate that taxol affects the interaction between protofilaments; to our knowledge, this is the first time that a ligand-binding site has been visualized in the tubulin molecule.