Mechanistic analysis of Kinesin-14 and Kinesin-2

Abstract

Drosophila melanogaster homodimeric kinesin-14 Ncd plays roles in spindle assembly and proper chromosome distribution through cross-linking parallel microtubules at the spindle poles and antiparallel microtubules within the spindle midzone. Like Saccharomyces cerevisiae kinesin-14 Kar3Vik1 and Kar3Cik1, which are heterodimers with one Kar3 catalytic motor domain and a Vik1/Cik1 partner motor homology domain , Ncd uses an ATP-promoted powerstroke mechanism and displays a similar microtubule binding configuration with two motor heads binding to adjacent microtubule protofilaments . Our hypothesis is that Kar3Vik1 and Kar3Cik1 as well as Ncd share a common ATPase mechanism for force generation although both Ncd heads can bind to nucleotides but Vik1/Cik1 cannot. To test this hypothesis, we performed presteady-state kinetics experiments and computational modeling, which established a common powerstroke model for Ncd. In this model, the ATPase mechanism for Ncd is very similar to those determined for Kar3Vik1 and Kar3Cik1, although microtubule interactions for these two heterodimers are not modulated by nucleotide state but by strain. Unlike conventional myosin-II or other processive molecular motors, two ATP turnovers are required for one powerstroke and microtubule-microtubule displacement. Furthermore, a slow step occurs after microtubule collision and before the ATP-promoted powerstroke. In summary, this model challenged the previous one head/one ATP turnover hypothesis and defined a common evolutionary mechanism for force generation of kinesin-14s from yeast to higher eukaryotes despite their structural and functional differences.

Unlike kinesin-14s, kinesin-2 motors are processive kinesins that can take over a hundred steps along a microtubule to deliver cargos in an anterograde movement. KIF3AC is one of the members of kinesin-2 family and contains two distinct motor polypeptides encoded by two different genes. KIF3AC is best known for its role in organelle transport in neurons. Our recent studies show that KIF3AC is as processive as conventional kinesin-1 and their ATPase mechanochemistry may be similar. However, the presence of two different motor polypeptides in KIF3AC implies that there must be a cellular advantage for the KIF3AC heterodimer. The hypothesis tested was whether there is an intrinsic bias within KIF3AC such that either KIF3A or KIF3C initiates the processive run. To pursue these experiments a mechanistic approach was used to compare the presteady-state kinetics of KIF3AC to the kinetics of homodimeric KIF3AA and KIF3CC. The results indicate that microtubule collision at 11.4 uM-1s-1 coupled with ADP release at 78 s-1 are fast steps for homodimeric KIF3AA. In contrast, KIF3CC exhibits much slower microtubule association at 2.1 uM-1s-1 and ADP release at 8 s-1. For KIF3AC, microtubule association at 6.6 uM-1s-1 and ADP release at 51 s-1 are intermediate between the constants for KIF3AA and KIF3CC that were engineered. These results indicate that either KIF3A or KIF3C can initiate the processive run. Surprisingly, the kinetics of the initial event of microtubule collision followed by ADP release for KIF3AC is not equivalent to 1:1 mixtures of KIF3AA plus KIF3CC homodimers at the same motor concentration. These results reveal that the intermolecular communication within the KIF3AC heterodimer modulates entry into the processive run regardless of whether the run is initiated by the KIF3A or KIF3C motor domain.

Kinesin-14 motors are referred to as C-kinesins because the motor domain is at the C-terminus, and they are involved in spindle organization and chromosome segregation in mitosis. In contrast, kinesin-2s are classified as N-kinesin motors with a motor domain at the N-terminus of polypeptides, and they are one of the major transporters in cells. Importantly, kinesin-14 motor proteins are unique in that they are nonprocessive, bind to adjacent microtubule protofilaments, and use a powerstroke mechanism to promote microtubule minus-end directed force generation, yet processive kinesin-2s step along a single protofilament and use an asymmetric hand-over-hand mechanism to generate microtubule plus-end directed force . The distinct mechanochemical mechanisms and microtubule binding patterns of these two kinesin motors are the major effectors contributing to their specific cellular functions.

Kinesins share a similar structure with two motor domains, C-terminal coiled-coil stalk and tail, yet each motor has a unique ATPase mechanism to generate force to fulfill cellular functions at the specific locations in diverse cell types. Kinesins use ATPase activity to generate microtubule-based functions, therefore, understanding the distinct mechanisms coupling chemical reactions with structural transitions is required to delineate the detailed contributions of individual kinesins in cells. The goal of this thesis is to define the mechanochemical features of two representative kinesins from different families, kinesin-14 and kinesin-2, which participate in distinct and specific cellular functions.