title: Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin.
abstract: 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.
authors.affiliation: Brain Development Research Group, Brain Science Institute, RIKEN, Wako, Saitama, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Uchimura
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Oguchi
authors.forename: Miho
authors.initials: M
authors.lastname: Katsuki
authors.forename: Takeo
authors.initials: T
authors.lastname: Usui
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Osada
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Nikawa
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Ishiwata
authors.forename: Etsuko
authors.initials: E
authors.lastname: Muto
mesh_terms: 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*
chemical_list: D050505:Mutant Proteins; D014404:Tubulin; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D016547:Kinesins