Microtubule (MT) binding accelerates the rate of ATP hydrolysis in kinesin. To understand the underlying mechanism, using charged-to-alanine mutational analysis, we identified two independent sites in tubulin, which are critical for kinesin motility, namely, a cluster of negatively charged residues spanning the helix 11-12 (H11-12) loop and H12 of alpha-tubulin, and the negatively charged residues in H12 of beta-tubulin. Mutation in the alpha-tubulin-binding site results in a deceleration of ATP hydrolysis (k(cat)), whereas mutation in the beta-tubulin-binding site lowers the affinity for MTs (K(0.5)MT). The residue E415 in alpha-tubulin seems to be important for coupling MT binding and ATPase activation, because the mutation at this site results in a drastic reduction in the overall rate of ATP hydrolysis, largely due to a deceleration in the reaction of ADP release. Our results suggest that kinesin binding at a region containing alpha-E415 could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Uchimura S, Oguchi Y, Hachikubo Y, Ishiwata S, Muto E

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