Chimeric Kinesin I/Eg5 Constructs Reveal Important Elements to Motor Activity

Abstract

Kinesin I (KHC) and kinesin V (Eg5) have very similar structure, yet very different roles. Eg5 is generally though of as having a low stall force (∼1.5pN), slow (unloaded velocity ∼100nm s−1), and having limited processivity, while KHC has a stall force of 5-7pN, a velocity of 500-700nm s−1, and having run lengths in the micron range. We have recently shown that the formation of the cover neck bundle (CNB), which is the formation of a beta sheet between B0 (the coverstrand) and B9 (the first half of the necklinker), is necessary for the motor to generate significant amounts of force. CNB formation along with docking of the necklinker to the motor head ( B7) creates the kinesin's power stroke. Loop 13 (L13), which has previously been shown to affect motor velocity with the mutation of highly conserved glycines to alanine, also forms contacts with B9, and has been shown to make a triple beta sheet structure consisting of B0, B9, and L13. To investigate the relative roles of the coverstrand, B9, and L13 in motor behavior we have created chimeric KHC/Eg5 constructs that incorporate the sequences for these elements from Eg5 into the KHC motorhead. We have found that stall force and unloaded run length are greatly affected by the substitution of Eg5 structural elements into KHC. These results suggest that the motors operate best with a matched CNB and that L13 strongly affects the mechanical strength of the motor. While a match CNB appears to make the relative motor function more robust, B9 has a larger impact on motor function than B0. Furthermore, these structural elements cause the motor to stall at lower forces, be slower, and less processive, but they alone do not turn KHC into Eg5.