ENGINEERED KLP61 F KINESIN CAN BE INHIBITED BY L5-DIRECTED COMPOUNDS: ALLOSTERIC MECHANISMS ARE CONSERVED ACROSS KINESIN-5 MOTORS
Document Type
Presentation
Start Date
22-10-2010 10:45 AM
End Date
22-10-2010 12:00 PM
Description
The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents, even amongst 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 (Klp61 F). Herein we ask whether or not drug sensitivity can be designed into Klp61 F. Two chimeric Klp61 F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response, comparable to HsEg5, in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61 F chimeras have de nova binding affinities for both STC and monastrol. 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. 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.
Recommended Citation
Liu, Liqiong; Parameswaran, S.; Liu, J.; and Kim, S., "ENGINEERED KLP61 F KINESIN CAN BE INHIBITED BY L5-DIRECTED COMPOUNDS: ALLOSTERIC MECHANISMS ARE CONSERVED ACROSS KINESIN-5 MOTORS" (2010). Dr. Joseph M. Moerschbaecher, III Graduate Research Day. 17.
https://digitalscholar.lsuhsc.edu/grad_rs/2010/poster1/17
ENGINEERED KLP61 F KINESIN CAN BE INHIBITED BY L5-DIRECTED COMPOUNDS: ALLOSTERIC MECHANISMS ARE CONSERVED ACROSS KINESIN-5 MOTORS
The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents, even amongst 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 (Klp61 F). Herein we ask whether or not drug sensitivity can be designed into Klp61 F. Two chimeric Klp61 F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response, comparable to HsEg5, in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61 F chimeras have de nova binding affinities for both STC and monastrol. 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. 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.