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

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Kinesin-1 walks along microtubules by alternately hydrolyzing ATP and moving two motor domains. Several recent studies have suggested that the strain developed through the neck linker is essential for the coordination between two motor domains, although the mechanism for the regulation of the motor domain's activity remains unknown. At the last annual meeting we employed disulfide-crosslinking between cysteine residues introduced into the neck linker and the motor domain to constrain the neck linker of a monomer in the forward or backward extended conformation, and using single molecule fluorescent observation we showed that detachment rate from the microtubule dramatically decreased when the neck linker is constrained in a backward extended conformation. To further investigate the effect of crosslinking on the ATP hydrolysis kinetics, we measured mant-ADP release after rapid mixing with microtubule and subsequent mant-ATP binding rates of the monomers. Stopped flow measurements showed that ADP release and ATP binding rates did not change after constraining in the backward extended conformation, suggesting that either ATP hydrolysis or Pi release step may be the rate-limiting. When the neck linker is constrained in a forward extended conformation, ATP binding rate did not changed but the ADP release rate dramatically decreased. These results suggest that ATP hydrolysis cycle of the motor domain can be differently regulated depending on the direction of the neck linker tension, explaining the alternate catalysis in the dimer.