Interaction of the mitotic inhibitor monastrol with human kinesin Eg5.
The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in the assembly of the mitotic spindle. It shows a three-domain structure with an N-terminal motor domain, a central coiled coil, and a C-terminal tail domain. In vivo HsEg5 is reversibly inhibited by monastrol, a small cell-permeable molecule that causes cells to be arrested in mitosis. Both monomeric and dimeric Eg5 constructs have been examined in order to define the minimal monastrol binding domain on HsEg5. NMR relaxation experiments show that monastrol interacts with all of the Eg5 constructs used in this study. Enzymatic techniques indicate that monastrol partially inhibits Eg5 ATPase activity by binding directly to the motor domain. The binding is noncompetitive with respect to microtubules, indicating that monastrol does not interfere with the formation of the motor-MT complex. The binding is not competitive with respect to ATP. Both enzymology and in vivo assays show that the S enantiomer of monastrol is more active than the R enantiomer and racemic monastrol. Stopped-flow fluorometry indicates that monastrol inhibits ADP release by forming an Eg5-ADP-monastrol ternary complex. Monastrol reversibly inhibits the motility of human Eg5. Monastrol has no inhibitory effect on the following members of the kinesin superfamily: MC5 (Drosophila melanogaster Ncd), HK379 (H. sapiens conventional kinesin), DKH392 (D. melanogaster conventional kinesin), BimC1-428 (Aspergillus nidulans BimC), Klp15 (Caenorhabditis elegans C-terminal motor), or Nkin460GST (Neurospora crassa conventional kinesin).
DeBonis
Salvatore
S
Institut de Biologie Structurale, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France.
Simorre
Jean-Pierre
JP
Crevel
Isabelle
I
Lebeau
Luc
L
Skoufias
Dimitrios A
DA
Blangy
Anne
A
Ebel
Christine
C
Gans
Pierre
P
Cross
Robert
R
Hackney
David D
DD
Wade
Richard H
RH
Kozielski
Frank
F
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004398:Dyneins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006131:Growth Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D008857:Microscopy, Interference; D018715:Microscopy, Video; D008870:Microtubules / Q000201:enzymology; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011743:Pyrimidines / Q000378:metabolism / Q000494:pharmacology*; D017384:Sequence Deletion; D012965:Sodium Chloride / Q000737:chemistry; D013237:Stereoisomerism; D013871:Thiones / Q000378:metabolism / Q000494:pharmacology*; D062367:ortho-Aminobenzoates / Q000737:chemistry
D006131:Growth Inhibitors; C470243:KIF11 protein, human; D010446:Peptide Fragments; D011743:Pyrimidines; D013871:Thiones; D062367:ortho-Aminobenzoates; D012965:Sodium Chloride; D000244:Adenosine Diphosphate; C400223:monastrol; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; D000255:Adenosine Triphosphate; D004398:Dyneins; D016547:Kinesins