Abstract The conserved kinesin-5 bipolar tetrameric motors slide apart microtubules during mitotic spindle assembly and elongation. Kinesin-5 bipolar organization originates from its conserved tetrameric helical minifilament, which position the C-terminal tail domains of two subunits near the N-terminal motor domains of two anti-parallel subunits (Scholey et al, 2014). This unique tetrameric structure enables kinesin-5 to simultaneously engage two microtubules and transmit forces between them, and for multiple kinesin-5 motors to organize via tail to motor interactions during microtubule sliding (Bodrug et al, 2020). Here, we show how these two structural adaptations, the kinesin-5 tail-motor domain interactions and the length of the tetrameric minifilament, determine critical aspects of kinesin-5 motility and sliding mechanisms. An x-ray structure of the 34-nm kinesin-5 minifilament reveals how the dual dimeric N-terminal coiled-coils emerge from the tetrameric central bundle. Using this structure, we generated active bipolar mini-tetrameric motors from Drosophila and human orthologs, which are half the length of native kinesin-5. Using single-molecule motility assays, we show that kinesin-5 tail domains promote mini-tetramers static pauses that punctuate processive motility. During such pauses, kinesin-5 mini-tetramers form multi-motor clusters mediated via tail to motor domain cross-interactions. These clusters undergo slow and highly processive motility and accumulate at microtubule plus-ends. In contrast to native kinesin-5, mini-tetramers require tail domains to initiate microtubule crosslinking. Although mini-tetramers are highly strained in initially aligning microtubules, they slide microtubules more efficiently than native kinesin-5, due to their decreased minifilament flexibility. Our studies reveal that the conserved kinesin-5 motor-tail mediated clustering and the length of the tetrameric minifilament are key features for sliding motility and are critical in organizing microtubules during mitotic spindle assembly and elongation.

The mechanochemical origins of the microtubule sliding motility within the kinesin-5 domain organization