Kar3Vik1 uses a minus-end directed powerstroke for movement along microtubules.
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.
The Boulder Laboratory for 3-D Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America.
Julia
J
Cope
Katherine C
KC
Rank
Susan P
SP
Gilbert
Ivan
I
Rayment
Andreas
A
Hoenger
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D020285:Cryoelectron Microscopy; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009068:Movement; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D012399:Rotation; D013269:Stochastic Processes
D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins