Key residues on microtubule responsible for activation of kinesin ATPase

Abstract

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 α-tubulin, and the negatively charged residues in H12 of Β-tubulin. Mutation in the α-tubulin- binding site results in a deceleration of ATP hydrolysis (k cat), whereas mutation in the Β-tubulin-binding site lowers the affinity for MTs (K 0.5 MT). The residue E415 in α-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 α-E415 could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP. © 2010 European Molecular Biology Organization | All Rights Reserved.