Optimal Size of the Neck Linker is Important for the Coordinated Processive Movement of Kinesin-1

Kinesin-1 is a dimeric motor protein that walks along microtubules by alternately moving two motor ‘heads’. Several recently published papers including ours provided evidences that kinesin dimer takes one-head-bound state while waiting for ATP and ATP-binding triggers the tethered head to bind to the forward tubulin-binding site. However, it is still not clear why rebinding of the tethered head, which is freely diffusing, to microtubule is prohibited during the ATP-waiting state. To explain this mechanism, we proposed a model based on the crystal structural analysis (Makino et al, this meeting) that ADP release of the tethered head is prohibited because the neck linker would be stretched out if both heads become nucleotide-free due to a steric hindrance posed on the neck linker. This model predicts that if the neck linker is artificially extended, the tethered head can easily rebind to the microtubule. To test this prediction, we engineered neck linker extended mutants by inserting poly-Gly residues and observed their conformational states using single-molecule FRET technique. We found that 5 amino acid extension of the neck linker allows the tethered head to rapidly rebind to the microtubule even in the absence of ATP, and that in this state both neck linkers adopt backward-pointing conformation. The neck linker extended mutants showed processive motility with reduced velocities compare to the wild-type, although the microtubule-activated ATPase rate was not changed, which are consistent with our previous results using poly-Pro insertion (Yildiz et al 2008). There results suggest that optimal size of the neck linker is important to prevent rebinding of the tethered head while waiting for ATP and to efficiently couple ATP hydrolysis energy with forward step.