Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin.
The kinesin-binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the alpha-helix 12 of beta-tubulin with alanine and analyzed their effect on kinesin-microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant. The unbinding force measurement revealed that in the former three mutants, the kinesin-microtubule interaction in the adenosine 5'-[beta,gamma-imido]triphosphate state (AMP-PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin-microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.
Uchimura
Seiichi
S
Brain Development Research Group, Brain Science Institute, RIKEN, Wako, Saitama, Japan.
Oguchi
Yusuke
Y
Katsuki
Miho
M
Usui
Takeo
T
Osada
Hiroyuki
H
Nikawa
Jun-ichi
J
Ishiwata
Shin'ichi
S
Muto
Etsuko
E
D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001696:Biomechanical Phenomena; D004252:DNA Mutational Analysis; D016547:Kinesins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D050505:Mutant Proteins; D014404:Tubulin; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D016547:Kinesins