Loop 5-directed Compounds Inhibit Chimeric Kinesin-5 Motors

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.The Kinesin-5 family of motor proteins plays a conserved role in the morphogenesis of the mitotic spindle. In particular, human Eg5 kinesin (HsEg5) 2 forms a homotetramer that is capable of cross-linking adjacent microtubule bundles during spindle formation and is essential for mitotic progression. High-throughput screens for small chemical inhibitors of mitosis have often yielded compounds that target this kinesin in particular. At the time of their discovery, these compounds represented the only known antimitotic compounds that did not directly affect cellular microtubule assembly or function and constituted an entirely new class of potential anticancer compounds. Important for our purposes, they also serve as tools to explore fundamental questions about motor protein function.To date, small chemical inhibitors have been discovered that bind to at least three different sites within the motor domain of HsEg5 (1-4). The most highly explored allosteric site is a single pocket whose absolute location was defined by xray crystallography (for examples, see . This site is formed by the ␣2 and ␣3 helices and capped by the L5 loop. This L5 pocket is on the surface of the motor domain and is ϳ12 Å and 22 Å from the nucleotide binding site and microtubule (MT)-binding site, respectively.Interactions of the L5 loop are at the crux of long distance, allosteric communication with the active site and the MTbinding site. The biochemical role of the L5 loop has been confirmed by kinetic measurements and mutagenesis efforts. Mutations throughout the loop result in varying degrees of inhibition of basal rates and MT-stimulated ATPase rates (3,7,(12)(13)(14)(15). Not limited in its effects upon the orthosteric site, a dihydropyrimidine derivative, monastrol, has been shown to affect h...

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

“…6B) explains why KLP61F is insensitive to drug inhibition (22,30). Drug sensitivity can be imposed on KLP61F if the human L5 sequence is spliced into its MD, emphasizing the unusual modularity of this loop (22).…”

“…Structures solved using x-ray crystallography have provided residue-by-residue views of the human K5 MD, including highlighting potential conformational variations in the K5 NL and L5 (11,15,20,21). In parallel, spectroscopic measurements have provided dynamic data concerning the timing and regulation of conformational changes within the MD and have been correlated with K5 enzyme kinetics (12,14,22). The K5 crystal structures have formed the basis of molecular interpretations of these dynamic data.…”

Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5

“…These control reactions established a noise baseline and never recorded a value in excess of 0.009 s Ϫ1 (average ϭ 0.004 Ϯ 0.002 s Ϫ1 (n ϭ 6)); control data are independent of, yet consistent with, prior laboratory publications (24,38). Data were analyzed using IGOR Pro software (Wavemetrics Inc.).…”

“…HsEg5 reaction plates contained an additional positive control sample. S-Trityl-L-cysteine, a well characterized and tightly binding inhibitor of HsEg5 (24,(35)(36)(37), was used to generate inhibited HsEg5 samples as a positive control, containing the complete reaction mixture, HsEg5 enzyme, and 100 M S-trityl-L-cysteine.…”

“…In addition, Kinesin-5 proteins house a druggable allosteric pocket that is conserved within the motor domain and yet variable in sequence across orthologs (20,24,25). Human Kinesin-5 inhibitors have been noted for their high degree of specificity for the target enzyme and lack of off-target effects (reviewed in Refs.…”