Article
- The EMBO Journal (2006) 25, 2263 - 2273
- doi:10.1038/sj.emboj.7601108
Published online: 27 April 2006
Subject Category:
A structural model for monastrol inhibition of dimeric kinesin Eg5
Troy C Krzysiak1,a, Thomas Wendt2,a, Lisa R Sproul1, Peter Tittmann3, Heinz Gross3, Susan P Gilbert1 and Andreas Hoenger2,b
- Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA
- European Molecular Biology Laboratory, Heidelberg, Germany
- Electron Microscopy ETH Zürich (EMEZ) c/o Institute for Applied Physics, Swiss Federal Technical High School, Zuerich-Hoenggerberg, Switzerland
Correspondence to:
Susan P Gilbert, Department of Biological Sciences, University of Pittsburgh, 518 Langley Hall, Pittsburgh, PA 15260, USA. Tel.: +1 412 624 5842; Fax: +1 412 624 4759; E-mail: spg1@pitt.edu
aThese authors contributed equally to this work
bPresent address: Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80302, USA
Received 4 November 2005; Accepted 31 March 2006
Abstract
Eg5 or KSP is a homotetrameric Kinesin-5 involved in centrosome separation and assembly of the bipolar mitotic spindle. Analytical gel filtration of purified protein and cryo-electron microscopy (cryo-EM) of unidirectional shadowed microtubule–Eg5 complexes have been used to identify the stable dimer Eg5-513. The motility assays show that Eg5-513 promotes robust plus-end-directed microtubule gliding at a rate similar to that of homotetrameric Eg5 in vitro. Eg5-513 exhibits slow ATP turnover, high affinity for ATP, and a weakened affinity for microtubules when compared to monomeric Eg5. We show here that the Eg5-513 dimer binds microtubules with both heads to two adjacent tubulin heterodimers along the same microtubule protofilament. Under all nucleotide conditions tested, there were no visible structural changes in the monomeric Eg5–microtubule complexes with monastrol treatment. In contrast, there was a substantial monastrol effect on dimeric Eg5-513, which reduced microtubule lattice decoration. Comparisons between the X-ray structures of Eg5-ADP and Eg5-ADP-monastrol with rat kinesin-ADP after docking them into cryo-EM 3-D scaffolds revealed structural evidence for the weaker microtubule–Eg5 interaction in the presence of monastrol.
Keywords:
- cryo-electron microscopy,
- KSP,
- microtubule,
- mitosis,
- molecular motor
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