Towards engineering artificial kinesin-14 motors with plus-end-directed processivity.

Abstract

Kinesins are motor proteins that utilize the chemical energy of ATP hydrolysis to generate movement and force inside cells on the microtubule filaments to play important roles in a variety of essential intracellular processes. Kinesin-14s are a subset of kinesins with C-terminal motor domains and commonly exhibit minus-end-directed motility on the microtubule. Our previous work revealed that KlpA, a kinesin-14 motor from the filamentous fungus Aspergillus nidulans, uniquely exhibits processive plus-end-directed motility on single microtubules as individual homodimers. We hypothesized that KlpA achieves plus-end-directed motility on a single microtubule by adopting a strained cis-configuration whereby the N-terminal tail and the C-terminal motor domains simultaneously bind to the same microtubule. To test this hypothesis, we combined bioinformatic analysis and in vitro single-molecule fluorescence microscopy to search for additional kinesin-14s that exhibit similar plus-end-directed motility as KlpA. We found that AnKinesin-14 from Aspergillus niger has a similar coiled-coil profile as that of KlpA and exhibits processive motility towards the microtubule plus ends (unpublished results). Additionally, we engineered a chimera that replaces the C-terminal motor domains of AnKinesin-14 with those of Ncd (a nonprecessive minus-end-directed kinesin-14) and retains the coiled-coil profile of AnKinesin-14. Our single-molecule motility experiments showed that the chimera moves processively on the microtubule towards the plus ends. Collectively, our findings not only provide strong support of our previous hypothesis for the plus-end-directed motility of KlpA but also lay out a conceptual framework for how to engineer artificial kinesin motors with desired directionality using a cut-and-paste approach.