Probing the Kinesin-Microtubule Interaction

Kinesin is a mechanoenzyme that couples adenosine triphosphate hydrolysis to the generation of force and movement along microtubules. To gain insight into the interactions of kinesin and microtubules, cross-linking, mapping, and proteolysis experiments were executed. The motor domain of kinesin was consistently cross-linked to both alpha- and beta-tubulin subunits. Initial mapping of the cross-linked kinesin suggested that amino acids within the N- and C-terminal cyanogen bromide fragments of the motor domain formed cross-links to both alpha- and beta-tubulin subunits. Mapping of the cross-linked tubulin suggested that cross-linking to kinesin motors occurred within the negatively charged, C-terminal cyanogen bromide fragments of alpha- and beta-tubulin subunits. Treatment of microtubules with subtilisin, a protease that cleaves C-terminal fragments from alpha- and beta-tubulin, reduced their ability to be cross-linked to kinesin motors supporting the idea that C-terminal sequences of alpha- and beta-tubulin may interact with kinesin motors. Finally, of three synthetic peptides, a peptide consisting of the last 12 C-terminal amino acids of beta-tubulin competitively interfered with the microtubule-stimulated adenosine triphosphatase activity of the kinesin motor, further suggesting that C-terminal sequences of beta-tubulin may be involved in kinesin binding.