Strain-Dependent Regulation of the Kinesin-1's Catalytic Activity as Studied by Disulfide-Crosslinking of the Neck Linker

Abstract

Kinesin is a dimeric motor protein that hydrolyzes ATP and moves along microtubules in a hand-over-hand manner. To walk by alternately moving two motor heads, the trailing head should detach from the microtubule prior to the leading head and the detached head should preferentially bind to the forward tubulin-binding site. To explain these mechanisms, we hypothesized that ATP hydrolysis reaction of kinesin motor domain can be regulated depending on the direction of the tension posed to the neck linker: backward strain posed to the neck linker suppresses ATP hydrolysis in the leading head and the forward strain posed to the neck linker suppresses ADP release at the trailing position. To test this hypothesis, we constrained the neck linker in the forward or backward extended conformation using disulfide-crosslinking between cysteine residues on the head and the neck linker, and examined these effects on the microtubule affinity and ADP release kinetics. Single molecule fluorescent observation of the GFP-fused monomeric kinesin showed that when the neck linker was constrained in a backward extended conformation, the dwell time on the microtubule in the presence of saturating ATP was increased by a factor of 15 compared to unconstrained condition. In contrast, stopped-flow measurement showed that when the neck linker was constrained in a forward extended conformation, ADP release rate after microtubule-binding was significantly decreased. These results support the idea that ATP hydrolysis cycle of kinesin's motor domain can be differently regulated depending on the direction of the neck linker extension.