Loop 5-directed compounds inhibit chimeric kinesin-5 motors: implications for conserved allosteric mechanisms.
The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents even among phylogenetic kin. For example, S-trityl-l-cysteine (STC) and monastrol are HsEg5 inhibitors that bind to a surface pocket created by the L5 loop, but neither compound inhibits the Drosophila Kinesin-5 homologue (Klp61F). Herein we ask whether or not drug sensitivity can be designed into Klp61F. Two chimeric Klp61F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response comparable with HsEg5 in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61F chimeras have de novo binding affinities for both STC and monastrol. These data show that the mechanism of intramolecular communication between the three ligand binding sites is conserved in the Kinesin-5 family, and reconstitution of a drug binding cassette within the L5 pocket is sufficient to restore allosteric inhibition. However, the two compounds were not equivalent in their allosteric inhibition. This surprising disparity in the response between the chimeras to monastrol and STC suggests that there is more than one allosteric communication network for these effectors.
Department of Biochemistry and Molecular Biology, LSU Health Sciences Center, New Orleans, Louisiana 70112, USA.
Liqiong
L
Liu
Sreeja
S
Parameswaran
Jing
J
Liu
Sunyoung
S
Kim
Edward J
EJ
Wojcik
D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013871:Thiones / Q000737:chemistry*
D029721:Drosophila Proteins; C084034:Klp61F protein, Drosophila; D008869:Microtubule-Associated Proteins; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D013871:Thiones; C003724:3-tritylthio-L-alanine; C400223:monastrol; D016547:Kinesins; D003545:Cysteine