Abstract
Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus [6]. However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Our results suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely to maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.