Key residues on microtubule responsible for activation of kinesin ATPase.
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.
Laboratory for Molecular Biophysics, Brain Science Institute, RIKEN, Wako, Saitama, Japan. uchimura@brain.riken.jp
Seiichi
S
Uchimura
Yusuke
Y
Oguchi
You
Y
Hachikubo
Shin'ichi
S
Ishiwata
Etsuko
E
Muto
D000595:Amino Acid Sequence; D001665:Binding Sites; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D016547:Kinesins