| Summary: | Kinesins are molecular motors that use energy from ATP hydrolysis to transport cargoes along microtubule tracks. There are at least 14 families of kinesins with different structural organisations but all kinesins have a motor domain that is the catalytic core for ATP hydrolysis and the binding site for microtubules. Most kinesins have a stalk domain, which facilitates oligomerisation, and a tail domain that is implicated in cargo binding and regulation. Depending on their structural organisation, each kinesin is suited for different functions. Some are involved in transporting vesicles and organelles in cells, while others are essential for axonal transport 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 kinesin that is made up of a dimer of anti-parallel dimers. By cross-linking anti-parallel microtubules and moving towards their plus ends, Eg5 slides them apart and establishes 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 that reason, Eg5 is a potential target for drug development in cancer chemotherapy with seven inhibitors in Phase I and II clinical trials. The first inhibitor of Eg5 was discovered 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 design approach. By determining crystal structures of the Eg5 motor domain in complex with various inhibitors, we can understand the interactions between the inhibitor and Eg5; thus, analysis of the structure-activity relationship (SAR) can help us to improve their potency. Consequently, these inhibitors could complement or act as alternatives to taxanes and vinca alkaloids, which are successful cancer chemotherapeutics currently used in the clinic, but have the tendency to cause neurotoxicities and develop resistance in patients. Here, I report the crystal structures of Eg5 in complex with three monastrol analogues, STLC, and four STLC analogues separately. Based on the crystal structures with monastrol analogues, I identified the preferential binding mode of each inhibitor and the main reasons for increased potency: namely the better fit of the ligand and the addition of two fluorine atoms. Next, the crystal structure of Eg5-STLC indicates that the three phenyl rings in STLC are buried in a mainly hydrophobic region, while the cysteine moiety of STLC is solventexposed. In addition, structures of Eg5 in complex with STLC analogues, which have meta- or para-substituents on one or more of the phenyl rings, reveal the positions of the substituents and provide valuable information for the SAR study. In short, these structures reveal important interactions in the inhibitor-binding pocket that will aid development of more potent inhibitors. To understand the molecular mechanism of inhibition, I examined the structure of the Eg5-STLC complex, which revealed an unprecedented intermediate state, whereby local changes at the inhibitor-binding pocket have not propagated to structural changes at the switch II cluster and neck linker. This provides structural evidence for the sequence of drug-induced conformational changes. In addition, I performed isothermal titration calorimetry to determine the thermodynamic parameters of the interaction between Eg5 and its inhibitors. The structural information and the thermodynamic parameters obtained help us to gain a better understanding of the molecular mechanism of inhibition by an Eg5 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. A prediction program showed that the first ~100 residues of the stalk domain have a high probability of forming a coiled-coil structure, while the middle ~150 residues have a low probability. Using analytical ultracentrifugation, I showed that the Eg5 stalk364-520 domain exists predominantly as a dimer with a sedimentation coefficient of 1.76 S. The purported coiled-coil quaternary structure is backed-up by circular dichroism data, which showed that Eg5 stalk364-520 domain contains about 52 % helical content. Finally, the low resolution solution structure of Eg5 stalk364-520 domain was determined by small angle X-ray scattering, which revealed an elongated structure that is ~165 Å in length. Together, these data give us a glimpse into the structural characteristics of the Eg5 stalk364-520 domain. Besides gaining a better understanding of Eg5, I decided to investigate the molecular mechanism of autoinhibition in conventional kinesin (later known as kinesin-1). As the founding member of kinesins, it was first discovered to be involved in axonal transport. When not transporting cargo, kinesin-1 is autoinhibited to prevent squandering of ATP. Although it is widely accepted that the tail binds to the motor domain to keep it in a folded autoinhibited state, the molecular mechanism remains unclear and several mechanisms have been proposed. Here, I report the crystal structures of the Drosophila melanogaster kinesin-1 motor domain dimer and the dimer-tail complex. The dimer, which exhibits ~180° rotational symmetry between the monomers, provides valuable structural information for modeling the motility of kinesins on microtubules. By comparing the free dimer with the dimer-tail complex, we observe that the motor domains have considerable freedom of movement in the absence of tail binding. However, in the dimer-tail complex, a ‘double lockdown’ at both the neck coil and the tail interface freezes out major movements. This could prevent conformational changes, such as neck linker undocking. Data from our collaborator (David Hackney) showed that a covalent cross-link, which mimics double lockdown of the dimer, prevents ADP release. Together, we propose a ‘double lockdown’ mechanism, in which cross-linking at both the coiled-coil and tail interface prevents the movement of the motor domains that is needed to undock the neck linker and release ADP. In short, the structures shed light on the autoinhibition mechanism, reveal important residues at the dimer-tail interface, invalidate other proposed mechanisms, and open up the possibility that other kinesins may be regulated by the same mechanism.
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