Common mechanistic themes for the powerstroke of kinesin-14 motors

Abstract

Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.

Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s

“…CryoEM and subsequent helical reconstruction of the MT complexes of Kar3Vik1 and Kar3Cik1 at different nucleotide states captured the same nucleotide-dependent powerstroke intermediates as reported for Ncd (10)(11)(12)(13)15). Apyrase was used to generate the nucleotide-free prepowerstroke state (Fig.…”

“…S1B). The publications documenting that Kar3Vik1, Kar3Cik1, and Ncd bind to adjacent protofilaments on the MT lattice (13,15) in combination with similarity in their prepowerstroke and postpowerstroke intermediates resulted in the experiments reported here to ask if homodimeric Ncd with two ATP binding sites can modulate MT interactions much like Kar3Vik1 and Kar3Cik1. The scheme in Fig.…”

“…Through our recent studies on S. cerevisiae Kar3Vik1 and Kar3Cik1, we discovered novel properties of these yeast kinesin14s that challenged the earlier models of how kinesin-14s generate force for their cellular functions (13,15,(26)(27)(28). The C-terminal globular domain of Vik1 exhibits the structure of a kinesin motor domain (MD), yet Vik1 as well as Cik1 lack a nucleotide-binding site (13,26).…”

“…In contrast to the microtubule (MT) plus-end directed processive kinesins, kinesin-14s are not processive as single molecules; they promote MT minus-enddirected force and use an ATP-promoted powerstroke to crosslink and slide one MT relative to another (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Sequence analysis indicates that all members of the kinesin-14 subfamily are dimeric, yet the structural organization of kinesin-14 motors differs from the N-terminal processive kinesins.…”

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation

“…one kinesin head results in turnover of one ATP molecule) for the motility of a single kinesin-14 motor (Endres et al, 2006;Gonzalez et al, 2013;Wendt et al, 2002), only one motor head needs to interact with microtubules and turnover of only one ATP molecule is necessary for the powerstroke, which combines both rotation and ATP turnover to slide the microtubules . However, a new ATP-promoted powerstroke hypothesis that implies two kinesin heads and turnover of two ATP molecules has been proposed for heterodimeric Kar3−Vik1 and Kar3−Cik1, as well as homodimeric Ncd−Ncd (Zhang et al, 2015).…”