A structural model for monastrol inhibition of dimeric kinesin Eg5.
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.
Krzysiak
Troy C
TC
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Wendt
Thomas
T
Sproul
Lisa R
LR
Tittmann
Peter
P
Gross
Heinz
H
Gilbert
Susan P
SP
Hoenger
Andreas
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020285:Cryoelectron Microscopy; D019281:Dimerization; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013871:Thiones / Q000737:chemistry / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
C470243:KIF11 protein, human; D011743:Pyrimidines; D013871:Thiones; D014404:Tubulin; D000244:Adenosine Diphosphate; C400223:monastrol; D000255:Adenosine Triphosphate; D016547:Kinesins