The Neck Linker of Kinesin-1 functions as a Regulator of ATP Hydrolysis Reaction

Abstract

Kinesin-1 is a highly processive motor that moves along microtubule in a hand-over-hand manner. The neck linker that connects two motor domains has been thought to act as a mechanical amplifier that propels the tethered head forward, however, we recently showed that the neck linker docking is not essential for the forward stepping (Isojima et al. this meeting). We hypothesized that the neck linker docking rather functions to activate ATP hydrolysis reaction. To test this hypothesis, we engineered various monomeric neck linker mutant kinesin and investigated the effect on the ATP hydrolysis reaction. As the neck linker was deleted further from the C-terminus, microtubule-activated ATPase rate was decreased and became almost undetectable when the whole neck linker was removed. The hydrophobic and complementary shaped side-chain of Ile325 is critical for promoting ATP hydrolysis reaction because substitution of this residue alone into glycine caused dramatic decrease in the ATPase rate. Single molecule fluorescent imaging showed that the Ile325G mutant monomer stably bound to the microtubule even in the presence of saturating ATP. A high-resolution cryo-electron microscopic study of the Ile325G mutant in complex with microtubule showed that the kinesin core is rotated clockwise direction even in the presence of saturating AMPPNP, which is similar to the conformation of wild type in the no-nucleotide state. These results suggest that Ile325 is essential for promoting ATP hydrolysis reaction by stabilizing counter clockwise rotation of kinesin motor domain upon ATP binding. This mechanism can also explain the front head gating mechanism for dimeric kinesin: the neck linker of the leading head is pulled backward so that the head cannot proceed ATP hydrolysis cycle until the trailing head detaches from microtubule.