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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 mec...

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Autores principales: Cope, Julia, Rank, Katherine C., Gilbert, Susan P., Rayment, Ivan, Hoenger, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3544905/
https://www.ncbi.nlm.nih.gov/pubmed/23342004
http://dx.doi.org/10.1371/journal.pone.0053792
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author Cope, Julia
Rank, Katherine C.
Gilbert, Susan P.
Rayment, Ivan
Hoenger, Andreas
author_facet Cope, Julia
Rank, Katherine C.
Gilbert, Susan P.
Rayment, Ivan
Hoenger, Andreas
author_sort Cope, Julia
collection PubMed
description 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.
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spelling pubmed-35449052013-01-22 Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules Cope, Julia Rank, Katherine C. Gilbert, Susan P. Rayment, Ivan Hoenger, Andreas PLoS One Research Article 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. Public Library of Science 2013-01-14 /pmc/articles/PMC3544905/ /pubmed/23342004 http://dx.doi.org/10.1371/journal.pone.0053792 Text en © 2013 Cope et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Cope, Julia
Rank, Katherine C.
Gilbert, Susan P.
Rayment, Ivan
Hoenger, Andreas
Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules
title Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules
title_full Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules
title_fullStr Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules
title_full_unstemmed Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules
title_short Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules
title_sort kar3vik1 uses a minus-end directed powerstroke for movement along microtubules
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3544905/
https://www.ncbi.nlm.nih.gov/pubmed/23342004
http://dx.doi.org/10.1371/journal.pone.0053792
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