| Abstract
| - The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in theassembly of the mitotic spindle. It shows a three-domain structure with an N-terminal motor domain, acentral coiled coil, and a C-terminal tail domain. In vivo HsEg5 is reversibly inhibited by monastrol, asmall cell-permeable molecule that causes cells to be arrested in mitosis. Both monomeric and dimericEg5 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 thisstudy. Enzymatic techniques indicate that monastrol partially inhibits Eg5 ATPase activity by bindingdirectly to the motor domain. The binding is noncompetitive with respect to microtubules, indicating thatmonastrol does not interfere with the formation of the motor−MT complex. The binding is not competitivewith respect to ATP. Both enzymology and in vivo assays show that the S enantiomer of monastrol ismore active than the R enantiomer and racemic monastrol. Stopped-flow fluorometry indicates thatmonastrol inhibits ADP release by forming an Eg5−ADP−monastrol ternary complex. Monastrol reversiblyinhibits the motility of human Eg5. Monastrol has no inhibitory effect on the following members of thekinesin 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).
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