Structural and functional characterisation ofconventional kinesin and mitotic kinesin Eg5 —a validated target for cancer chemotherapy

[摘要] Kinesins are molecular motors that use energy from ATP hydrolysis to transportcargoes along microtubule tracks. There are at least 14 families of kinesins withdifferent structural organisations but all kinesins have a motor domain that isthe catalytic core for ATP hydrolysis and the binding site for microtubules. Mostkinesins have a stalk domain, which facilitates oligomerisation, and a tail domainthat is implicated in cargo binding and regulation. Depending on their structuralorganisation, each kinesin is suited for different functions. Some are involved intransporting vesicles and organelles in cells, while others are essential for axonaltransport in neurons. Still others are involved in intraflagellar transport in cilia.Lastly, a group of kinesins participate in different steps of mitosis.One such kinesin is the human mitotic kinesin Eg5. It is a homotetrameric kinesinthat is made up of a dimer of anti-parallel dimers. By cross-linking anti-parallelmicrotubules and moving towards their plus ends, Eg5 slides them apart andestablishes the bipolar spindle. When Eg5 is inhibited by antibodies or siRNA,cells arrest in mitosis with non-separated centrosomes and monoastral spindles.Prolonged mitotic arrest eventually leads to apoptotic cell death. For thatreason, Eg5 is a potential target for drug development in cancer chemotherapywith seven inhibitors in Phase I and II clinical trials. The first inhibitor of Eg5 wasdiscovered in a phenotype-based screen and is called monastrol. Since then,several classes of inhibitors, such as ispinesib (a clinical trial candidate) and Strityl-L-cysteine (STLC), have been discovered.To develop more potent inhibitors, we employed a structure-based drug designapproach. By determining crystal structures of the Eg5 motor domain in complexwith various inhibitors, we can understand the interactions between theinhibitor and Eg5; thus, analysis of the structure-activity relationship (SAR) canhelp us to improve their potency. Consequently, these inhibitors couldcomplement or act as alternatives to taxanes and vinca alkaloids, which aresuccessful cancer chemotherapeutics currently used in the clinic, but have thetendency to cause neurotoxicities and develop resistance in patients.Here, I report the crystal structures of Eg5 in complex with three monastrolanalogues, STLC, and four STLC analogues separately. Based on the crystalstructures with monastrol analogues, I identified the preferential binding modeof each inhibitor and the main reasons for increased potency: namely the betterfit of the ligand and the addition of two fluorine atoms. Next, the crystalstructure of Eg5-STLC indicates that the three phenyl rings in STLC are buried ina mainly hydrophobic region, while the cysteine moiety of STLC is solventexposed.In addition, structures of Eg5 in complex with STLC analogues, whichhave meta- or para-substituents on one or more of the phenyl rings, reveal thepositions of the substituents and provide valuable information for the SAR study.In short, these structures reveal important interactions in the inhibitor-bindingpocket that will aid development of more potent inhibitors.To understand the molecular mechanism of inhibition, I examined the structureof the Eg5-STLC complex, which revealed an unprecedented intermediate state,whereby local changes at the inhibitor-binding pocket have not propagated tostructural changes at the switch II cluster and neck linker. This providesstructural evidence for the sequence of drug-induced conformational changes. Inaddition, I performed isothermal titration calorimetry to determine thethermodynamic parameters of the interaction between Eg5 and its inhibitors.The structural information and the thermodynamic parameters obtained help usto gain a better understanding of the molecular mechanism of inhibition by anEg5 inhibitor.While there is a large amount of information about the motor domain of Eg5,less is known about the stalk domain, which facilitates oligomerisation. Aprediction program showed that the first ~100 residues of the stalk domain havea high probability of forming a coiled-coil structure, while the middle ~150residues have a low probability. Using analytical ultracentrifugation, I showedthat the Eg5 stalk364-520 domain exists predominantly as a dimer with asedimentation coefficient of 1.76 S. The purported coiled-coil quaternarystructure is backed-up by circular dichroism data, which showed that Eg5stalk364-520 domain contains about 52 % helical content. Finally, the low resolutionsolution structure of Eg5 stalk364-520 domain was determined by small angle X-rayscattering, which revealed an elongated structure that is ~165 Å in length.Together, these data give us a glimpse into the structural characteristics of theEg5 stalk364-520 domain.Besides gaining a better understanding of Eg5, I decided to investigate themolecular mechanism of autoinhibition in conventional kinesin (later known askinesin-1). As the founding member of kinesins, it was first discovered to beinvolved in axonal transport. When not transporting cargo, kinesin-1 isautoinhibited to prevent squandering of ATP. Although it is widely accepted thatthe tail binds to the motor domain to keep it in a folded autoinhibited state, themolecular mechanism remains unclear and several mechanisms have beenproposed. Here, I report the crystal structures of the Drosophila melanogasterkinesin-1 motor domain dimer and the dimer-tail complex. The dimer, whichexhibits ~180° rotational symmetry between the monomers, provides valuablestructural information for modeling the motility of kinesins on microtubules.By comparing the free dimer with the dimer-tail complex, we observe that themotor domains have considerable freedom of movement in the absence of tailbinding. However, in the dimer-tail complex, a ‘double lockdown’ at both theneck coil and the tail interface freezes out major movements. This couldprevent conformational changes, such as neck linker undocking. Data from ourcollaborator (David Hackney) showed that a covalent cross-link, which mimicsdouble lockdown of the dimer, prevents ADP release. Together, we propose a‘double lockdown’ mechanism, in which cross-linking at both the coiled-coil andtail interface prevents the movement of the motor domains that is needed toundock the neck linker and release ADP. In short, the structures shed light onthe autoinhibition mechanism, reveal important residues at the dimer-tailinterface, invalidate other proposed mechanisms, and open up the possibilitythat other kinesins may be regulated by the same mechanism.

[发布机构] University:University of Glasgow;Department:Institute of Cancer Sciences

[关键词] Kinesin, molecular motor, cancer, autoinhibition, structural biology