Nucleotide binding and hydrolysis induces a disorder-order transition in the kinesin neck-linker region.

Kinesin translocation is thought to occur by a conformational change in a region of the motor domain called the neck linker. However, most evidence supporting this hypothesis comes from monomeric constructs unable to move processively. To address this issue, we investigated the neck-linker configuration on microtubule-bound monomeric and dimeric kinesin constructs using single-molecule fluorescence polarization microscopy. We found that the neck-linker region (i) is very mobile in the absence of nucleotides and during steady walking, (ii) decreases mobility and aligns along the microtubule axis in the presence of AMPPNP or ADP + AlF4(-), (iii) is mostly ordered in the monomeric constructs in the presence of ADP + AlF4(-), and (iv) is closer to parallel to the microtubule axis in the dimeric constructs. These results support the proposed role of the neck linker and suggest a coordination mechanism between the two motor domains in the dimer.