Structure of tubulin at 6.5 A and location of the taxol-binding site.
Tubulin, the major component of microtubules, is a heterodimer of two chains, alpha and beta, both of relative molecular mass 50,000 (Mr50K) and with 40-50% identity. The isotypic variety and conformational flexibility of tubulin have so far made it impossible to obtain crystals for X-ray work. Structural knowledge of tubulin has been limited to about 20 A from X-ray diffraction of oriented microtubules, and from electron microscopy of microtubules and zinc-induced crystalline sheets in negative stain. The sheets consist of protofilaments similar to those in microtubules but associated in an antiparallel arrangement, and their two-dimensional character is ideal for high-resolution electron microscopy. Here we present a three-dimensional reconstruction of tubulin to 6.5 A resolution, obtained by electron crystallography of zinc-induced two-dimensional crystals of the protein. The alpha- and beta-subunits appear topologically similar, in agreement with their sequence homology. Several features can be defined in terms of secondary structure. An apparent alpha-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 alpha- and beta-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 microtubules. 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.
Nogales
E
E
Life Science Division, Lawrence Berkeley Laboratory, California 94720, USA.
Wolf
S G
SG
Khan
I A
IA
Ludueña
R F
RF
Downing
K H
KH
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D017239:Paclitaxel / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D014404:Tubulin; D017239:Paclitaxel