Abstract
Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.
Highlights
Microtubules (MTs) form tracks for the active transport of vesicles and macromolecules inside eukaryotic cells, generate pulling forces during assembly of mitotic spindles, and promote the alignment and segregation of chromosomes (Goshima and Scholey, 2010; Vale, 2003)
To better understand the role of the tail domain on kinesin-5 function, we first evaluated the effect of the tail domain on MT-stimulated ATP hydrolysis by the kinesin-5 motor domain
The addition of equimolar KLP61F tail to a mixture of the motor and MTs led to a two-fold decrease in kcat (3.5 sÀ1 vs 7.1 sÀ1) but produced little change in Km (756 nM vs 680 nM) suggesting that the tail does not competitively interfere with motor-MT binding but rather modulates MT-stimulated ATP hydrolysis (Figure 1C)
Summary
Microtubules (MTs) form tracks for the active transport of vesicles and macromolecules inside eukaryotic cells, generate pulling forces during assembly of mitotic spindles, and promote the alignment and segregation of chromosomes (Goshima and Scholey, 2010; Vale, 2003). The bipolar tetrameric organization of the kinesin-5 BASS region orients the two-parallel coiled-coils at the neck and their associated motor domains at an off-set This results in a 100 ̊-lateral rotation between the two opposite ends that potentially mediates the preference for kinesin-5 to bind and slide two antiparallel MTs (Scholey et al, 2014). Tail-deletion leads to a loss of human Eg5 mitotic spindle localization in mammalian cells while retaining MT binding capability These studies suggest a revised model for kinesin-5mediated MT sliding in which the tail domain slows MT-stimulated ATP hydrolysis at each end of the homotetramer and enhance force production essential for MT sliding
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