Processive Motility of Heterodimeric Kinesin That Has Defect in the Neck Linker Docking

Abstract

Kinesin-1 is a motor protein that moves processively along microtubules in a hand-over-hand manner. The neck linker, a short stretch that connects two motor heads, has been shown to undergo ATP-dependent conformational changes, although its role on the processive motility is still controversial. To address this question, we employed a kinesin mutant at switch I (R203K) that can normally bind ATP but is unable to hydrolyse ATP (Klumpp et al. JBC 2003). First we observed the neck linker structural state of monomeric R203K bound to the microtubule using single molecule FRET. We found that the neck linker remained undocked conformation even in the presence of saturating ATP, suggesting that ATP-binding is not sufficient to stabilize neck linker docked state. Next we constructed heterodimeric kinesin that is composed of a wild-type head and a mutant R203K head. As recently been shown by Thoresen and Gelles (Biochemistry 2008), this heterodimer showed slow processive movement along microtubules. Then we observed the conformational changes of this heterodimer using a single molecule FRET sensor as previously developed to distinguish one-head-bound and two-head-bound states (Mori et al. Nature 2007), and found that the heterodimer showed hand-over-hand movement. Unexpectedly, they spent most of the time in the two-head-bound state where wild-type head is in the front and the mutant head is in the rear, indicating that the displacement of rear wild-type head to the forward binding site is not the rate-limiting. These results suggest that the neck linker docking in the microtubule-bound head is not essential for the tethered head to translate and bind to the forward tubulin-binding site and rather is required for promoting ATP-hydrolysis and subsequent detachment of the trailing head from microtubule.