UC Berkeley

Kinesin is a molecular motor that walks on microtubules by taking 8 nm steps towards the plus end in a hand over hand manner. The processive motility of kinesin requires its two motor domains (heads) to coordinate with each other. Interhead coordination involves a gating mechanism, where one of the heads cannot proceed until the other head goes through a certain act. Recent studies suggested that neck linker orientation is involved in coordination of the nucleotide binding and stepping motion of the two heads. In the leading head, the neck-linker is pointing backward, and inhibiting nucleotide binding. In the trailing head, the neck-linker is pointing forward, which allows the release of hydrolysis products and stepping towards the next tubulin binding site. To test this hypothesis, I used a novel optical trapping assay to directly measure microtubule release rate of the motor under a large range of constant forces. In the absence of nucleotide, the microtubule release increased as a function of force in both directions, with the release towards the plus-end was slightly faster than the minus-end under the same force. Addition of ATP resulted in faster release in both directions, consistent with nucleotide induced release properties of the kinesin motor. When kinesin was pulled from its linker in the forward direction, we observed faster release in the presence of ADP, which mimics the microtubule release. Remarkably, release was relatively slow when the linker was oriented in the backward direction by trap. Such asymmetry in the presence of ADP was not observed when kinesin was pulled from its head by a short DNA tether. These results suggest that geometrical constraints of the neck linker domains in a walking kinesin dimer break the symmetry of the two identical heads, such that the trailing head is free to move when the leading head is unable to bind nucleotide.