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 pivotal role in the head-head coordination but its exact role is still controversial. It have been widely believed that the neck linker acts as a mechanical element to propel the tethered head forward, however, we recently proposed an alternative model (biased-capturing model) based on crystallographic and cryo-EM analyses, in which the neck linker docking is not required for the forward stepping. We hypothesized that the neck linker docking rather functions to activate rate-limiting ATP hydrolysis reaction.To test this hypothesis, we engineered a series of monomeric kinesin mutants whose neck linker was truncated and carried out biochemical and structural analyses. As the neck linker was deleted further from the C-terminus, microtubule-activated ATPase rate of the mutant kinesin decreased and it becomes almost undetectable when whole neck linker was removed. Single molecule fluorescent imaging showed that the neck linker-less monomer stably bound to the microtubule even in the presence of 1 mM ATP. Cryo-EM observation of the neck linker-less mutant on the microtubule in the presence of saturating AMP-PNP displayed a structure similar to that of nucleotide-free wild-type kinesin.These results indicate that kinesin without the neck linker can bind to the microtubule but is incapable of proceeding ATP hydrolysis reaction, which is consistent with the idea that the neck linker acts as an activator of ATP hydrolysis reaction. This mechanism can explain the front head gating mechanism for head-head coordination: the neck linker of the leading head is pulled backward and the head cannot proceed ATP hydrolysis so that the head cannot detach until the trailing head detaches from microtubule.