A Structural Model of the Kinesin-5 Mechanochemical Cycle

Abstract

Kinesin motor domains drive diverse microtubule-based, ATP-dependent activities but the molecular adaptations that specify these diverse functions are poorly understood. Kinesin-5s are essential mitotic motors and their inhibition with specific small molecules blocks cell division. Using cryo-electron microscopy and subnanometer resolution structure determination, we have visualised conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. In the ATP-like state, the kinesin-5 neck-linker is directed towards the microtubule plus-end, consistent with its role in directional force generation. As ATP hydrolysis proceeds, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain, uncurling of the drug-binding loop5 and discrete, ratchet-like displacements of the neck linker that contribute to motor stepping. The motor N-terminus also undergoes large reorientations that indicate its role in controlling kinesin-5 neck-linker conformation throughout the motor's ATPase cycle. A kinesin-5 mutant lacking this N-terminus is enzymatically active, but ATP-dependent neck linker movement and motility is defective, although not totally ablated. Our data demonstrate that, while the motor N-terminus plays a kinetic role in controlling efficient neck-linker movement, the kinesin-5 neck-linker has intrinsic biophysical properties that enable it undergo nucleotide-dependent ratchet-like movements that have presumably evolved according to specific functional requirements.