Abstract
We have used cryo-electron microscopy (cryo-EM) and helical averaging to examine the 3-D structure of the heterodimeric kinesin-14 Kar3Vik1 complexed to microtubules at a resolution of 2.5 nm. 3-D maps were obtained at key points in Kar3Vik1’s nucleotide hydrolysis cycle to gain insight into the mechanism that this motor uses for retrograde motility. In all states where Kar3Vik1 maintained a strong interaction with the microtubule, we found, as observed by cryo-EM, that the motor bound with one head domain while the second head extended outwards. 3-D reconstructions of Kar3Vik1-microtubule complexes revealed that in the nucleotide-free state, the motor’s coiled-coil stalk points toward the plus-end of the microtubule. In the ATP-state, the outer head is shown to undergo a large rotation that reorients the stalk ∼75° to point toward the microtubule minus-end. To determine which of the two heads binds to tubulin in each nucleotide state, we employed specific Nanogold®-labeling of Vik1. The resulting maps confirmed that in the nucleotide-free, ATP and ADP+Pi states, Kar3 maintains contact with the microtubule surface, while Vik1 extends away from the microtubule and tracks with the coiled-coil as it rotates towards the microtubule minus-end. While many previous investigations have focused on the mechanisms of homodimeric kinesins, this work presents the first comprehensive study of the powerstroke of a heterodimeric kinesin. The stalk rotation shown here for Kar3Vik1 is highly reminiscent of that reported for the homodimeric kinesin-14 Ncd, emphasizing the conservation of a mechanism for minus-end directed motility.
Highlights
The microtubule (MT)-associated motor, Kar3 [1] is the motor domain of a minus-end directed kinesin-14 with important roles in mitosis and karyogamy in Saccharomyces cerevisiae
Our results reveal that Kar3Vik1 uses a powerstroke reminiscent of that described for Ncd, highlighting the conservation of a mechanism of movement among homo- and heterodimeric kinesin-14 motors
Stochastic binding of Kar3MD to MTs has been reported previously by immunofluorescence, but here we employed electron microscopy to visualize individual Kar3MDs bound to the MT lattice
Summary
The microtubule (MT)-associated motor, Kar3 [1] is the motor domain of a minus-end directed kinesin-14 with important roles in mitosis and karyogamy in Saccharomyces cerevisiae. Numerous structural studies have been conducted by cryo-EM 3-D analysis of the kinesin-microtubule interaction in the strong binding states, typically the nucleotide-free state (generated using the ATP- and ADP-hydrolyzing enzyme, apyrase), and the ATP state (mimicked with the non-hydrolyzable ATP analog, AMPPNP). We have employed cryo-EM and helical 3-D analysis of MTs decorated with a heterodimeric Kar3Vik1 [6] to analyze the relevant structural changes that occur within the heterodimer in response to changes in nucleotide state With these methods we were able to directly visualize the powerstroke responsible for generating Kar3Vik1’s retrograde motility. We designed a ‘‘cys-light’’ Kar3Vik construct with only one cysteine located on the C-terminal motor homology domain of Vik1 By labeling this cysteine residue with a 1.4 nm maleimide-NanogoldH particle, we could show that for the nucleotide-free, ATP, and ADP+Pi states Kar maintains contact with the MT while Vik remains tethered. Our results reveal that Kar3Vik uses a powerstroke reminiscent of that described for Ncd, highlighting the conservation of a mechanism of movement among homo- and heterodimeric kinesin-14 motors
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