Flexibility of the neck-linker during docking is pivotal for function of bi directional kinesin

The role of the neck-linker (NL) element in regulating the functions of bi-directional kinesins is unknown. We report that replacing the NL of the bi-directional kinesin-5 Cin8 with sequences from plus-end directed kinesins produces non-functional Cin8 with defective spindle localization and abolished minus-end directionality and microtubule-crosslinking in vitro. Mutation of a single glycine in the NL of Cin8 to asparagine (proposed to serve as an N-latch that stabilizes the docked conformation of the NL in the plus-end directed kinesins) causes defects in the functions of Cin8. Strikingly, in a non-functional Cin8 containing the NL of the plus-end directed kinesin-5 Eg5, a single mutation of the N-latch asparagine back to glycine rescues the in vivo and in vitro defects. Since such replacement eliminates stabilizing interactions between the docked NL and the motor domain, we conclude that flexibility of NL during docking is pivotal for the function of bi-directional kinesin motors.