Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains

Abstract

STUDIES of immobilized kinesin have shown that a single dimeric molecule can maintain contact with and drive sliding of a microtubule^1,2. In solution, however, native kinesin binds micro-tubules too weakly³ and hydrolyses ATP too slowly to produce the high sliding velocities seen in motility assays⁴. This apparent inhibition in solution appears to be caused by the binding of kines-in's tail domains to its motor (head) domains in a folded conformation⁵. DKH392, a construct containing two heads but no tails, has been shown to display both tight binding to microtubules and high ATPase rates⁶. Furthermore, it retains one molecule of ADP per dimer when bound to microtubules⁷, which could facilitate a 'hand-over-hand' mechanism for processive motion. Here we show that DKH392 hydrolyses more than 100 ATP molecules per diffusional encounter with a microtubule, even in the high-salt conditions encountered physiologically. This provides direct evidence that kinesin's activity is highly processive, with the motor remaining attached to a microtubule through many cycles of ATP hydrolysis.