Forward Stepping Mechanism of Kinesin-1 Studied using Asymmetrically-Joined Two-Headed Monomer

Abstract

Kinesin-1 moves processively along microtubule by alternately moving two motor domains, but the mechanism of the preferential forward stepping is still controversial. The “neck linker-docking model” proposes that the neck linker docking of the microtubule-bound head generates forward bias of the tethered head. However, our recent structural analysis of kinesin dimer (Makino et al.) suggested an alternate model in which the tethered head position does not necessarily be biased because the tethered head is not allowed to bind to the rear tubulin-binding site due to a steric constraint on its neck linker and can only release ADP at the forward binding site (“biased-binding model”). To distinguish these mechanisms as alternate steps, we engineered two-headed monomer kinesin by joining two motor heads in tandem on a single polypeptide, in which the neck linker of first head (N-head) is connected to second head (C-head) so that it can propel C-head forward, whereas the neck linker of C-head is free. Single molecule fluorescence observation showed that this two-headed monomer moves processively along microtubules although the velocity was smaller than wild-type dimer by four-fold. In addition, FIONA measurement of individual head showed that both heads takes discrete 16 nm steps, illustrating that this monomer moves by alternately exchanging two heads. Then we measured the dwell time of alternate steps using single molecule FRET and found that forward-stepping of C-head presumably driven by the neck linker docking was less efficient than the forward-stepping of N-head, because the tethered C-head often rebinds to the rear-binding site. These results suggest that biased-binding mechanism is more efficient to drive forward stepping, because rebinding of the tethered head to the rear-binding site is effectively prohibited.