Loop 5-directed Compounds Inhibit Chimeric Kinesin-5 Motors: IMPLICATIONS FOR CONSERVED ALLOSTERIC MECHANISMS

Liqiong Liu,Sreeja Parameswaran,Jing Liu,Sunyoung Kim,Edward J Wojcik

doi:10.1074/jbc.m110.154989

Liqiong Liu, Sreeja Parameswaran + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.154989

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Abstract

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.

Highlights

The Kinesin-5 family of motor proteins plays a conserved role in the morphogenesis of the mitotic spindle
To design the L5 pocket Klp61F chimeras, we examined the residues in the L5 pocket of HsEg5 co-crystallized with either STC or monastrol
To determine whether Klp61F is refractory to allosteric inhibition due to limited effector binding activity, we explored whether these effectors exhibit measurable affinity to wild type Klp61F by isothermal titration calorimetry (ITC)

Summary

Introduction

The Kinesin-5 family of motor proteins plays a conserved role in the morphogenesis of the mitotic spindle. The most highly explored allosteric site is a single pocket whose absolute location was defined by xray crystallography (for examples, see Ref. 5–11). This site is formed by the ␣2 and ␣3 helices and capped by the L5 loop. There are still outstanding unanswered questions regarding this allosteric site in Kinesin-5 proteins It remains unclear how the L5 loop transmits the inhibitory signal or conformational change that affects the orthosteric and MT-binding sites. In crystallographic studies of HsEg5-ADP complexed with monastrol [7, 10] and other L5 pocket inhibitors (for example, Ref. 19), the wild type Kinesin-5 motor domain displays a similar conformer irrespective of the chemical nature of the allosteric drug. Regardless, examination of co-crystal structures of drugs complexed with HsEg5 do not reveal any pronounced perturbation of the active site residues, such as those of the P-loop, or Mg2ϩ cofactor that might explain the aborted catalytic cycle

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