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

Abstract

Drosophila melanogaster kinesin-14 Ncd cross-links parallel microtubules at the spindle poles and antiparallel microtubules within the spindle midzone to play roles in bipolar spindle assembly and proper chromosome distribution. As observed for Saccharomyces cerevisiae kinesin-14 Kar3Vik1 and Kar3Cik1, Ncd binds adjacent microtubule protofilaments in a novel microtubule binding configuration and uses an ATP-promoted powerstroke mechanism. The hypothesis tested here is that Kar3Vik1 and Kar3Cik1, as well as Ncd, use a common ATPase mechanism for force generation even though the microtubule interactions for both Ncd heads are modulated by nucleotide state. The presteady-state kinetics and computational modeling establish an ATPase mechanism for a powerstroke model of Ncd that is very similar to those determined for Kar3Vik1 and Kar3-Cik1, although these heterodimers have one Kar3 catalytic motor domain and a Vik1/Cik1 partner motor homology domain whose interactions with microtubules are not modulated by nucleotide state but by strain. The results indicate that both Ncd motor heads bind the microtubule lattice; two ATP binding and hydrolysis events are required for each powerstroke; and a slow step occurs after microtubule collision and before the ATP-promoted powerstroke. Note that unlike conventional myosin-II or other processive molecular motors, Ncd requires two ATP turnovers rather than one for a single powerstroke-driven displacement or step. These results are significant because all metazoan kinesin-14s are homodimers, and the results presented show that despite their structural and functional differences, the heterodimeric and homodimeric kinesin-14s share a common evolutionary structural and mechanochemical mechanism for force generation.presteady-state kinetics | dynamic modeling | microtubules I n the early stages of mitosis and meiosis, the bipolar metaphase spindle must be established, and kinesin-14 molecular motors play key roles in this process (1-4). 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 (5-17). 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. The kinesin-14s exhibit C-terminal motor domains connected by an N-terminal continuous coiled-coil stalk with an N-terminal ATP-independent MT binding site (7,(18)(19)(20)(21)(22). And although most of the kinesin-14s are homodimeric, some yeast species, including Saccharomyces cerevisiae and Candida glabrata, contain heterodimeric kinesin-14s (13,14,19). The conventional hypothesis for homodimeric kinesin-14 force generation proposed that only one motor head interacts with the MT and only one ATP turnover is required to complete the powerstroke (Fig. S1A) (10, 12). In contrast, our presteady-state kinetics (9,...

Fast or Slow, Either Head Can Start the Processive Run of Kinesin-2 KIF3AC

“…Pulse-Chase Kinetics of ATP Binding-To determine the time course of ATP binding, a chemical quench-flow instrument (RQF-3, KinTek Corp.) was used (32,33). The MT⅐KIF3AC complex (5 M KIF3AC/40 M MTs or 10 M KIF3AC/40 M MTs, syringe concentrations) was rapidly mixed with MgATP plus radiolabeled [␣-32 P]ATP and 300 mM KCl (syringe concentrations) for times ranging from 5 to 200 ms in the instrument.…”

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

“…Mitotic spindles are composed of three main types of dynamic microtubule: (i) astral microtubules that are generated from the MTOC and extending towards the cell cortex (Reinsch and Gönczy, 1998); (ii) interpolar microtubules that overlap at the midzone with antiparallel orientation (Cai et al, 2009b;Hentrich and Surrey, 2010); (iii) kinetochore fibers (K-fibers) that are generated by centrosome capture through the kinetochore (Hayden et al, 1990;Maiato et al, 2004 corresponds to approximately two to six 8-nm steps (Foster and Gilbert, 2000;Zhang et al, 2015). In the same study, a hold-andrelease mechanism for kinesin-14 motors was proposed in contrast to the hand-over-hand model established for conventional kinesins (Zhang et al, 2015).…”

“…In the same study, a hold-andrelease mechanism for kinesin-14 motors was proposed in contrast to the hand-over-hand model established for conventional kinesins (Zhang et al, 2015). To date, three main means used by kinesin-14 motors to generate weakly processive movements have been described (Fig.…”

“…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). The interactions between both Ncd heads and microtubules depend on the nucleotide state but interactions between either Vik1 or Cik1 and microtubules are regulated by strain (Zhang et al, 2015).…”

“…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). The interactions between both Ncd heads and microtubules depend on the nucleotide state but interactions between either Vik1 or Cik1 and microtubules are regulated by strain (Zhang et al, 2015). Furthermore, turnover of two ATP molecules is required for interaction with microtubules as well as force generation in the case of the kinesin-14 motors that bind to the adjacent microtubule lattices (Endres et al, 2006;Gonzalez et al, 2013;Kocik et al, 2009;Zhang et al, 2015).…”