The dimeric form of the kinesin motor and neck domain from rat brain with bound ADP has been solved by X-ray crystallography. The two heads of the dimer are connected via a coiled-coil alpha-helical interaction of their necks. They are broadly similar to one another; differences are most apparent in the head-neck junction and in a moderate reorientation of the neck helices in order to adopt to the coiled-coil conformation. The heads show a rotational symmetry (approximately 120 degrees) about an axis close to that of the coiled-coil. This arrangement is unexpected since it is not compatible with the microtubule lattice. In this arrangement, the two heads of a kinesin dimer could not have equivalent interactions with microtubules.
In cells, stable microtubules are covalently modified by a carboxy-peptidase which removes the C-terminal tyrosine residue of α-tubulin. The significance of this selective detyrosination of microtubules is not understood. Here, we report that tubulin detyrosination in fibroblasts inhibits microtubule disassembly. This inhibition is relieved by overexpression of the depolymerizing motor MCAK. Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated microtubules in fibroblasts. Detyrosination of microtubules suppresses the activity of MCAK in vitro, apparently due to a decreased affinity of the ADP-Pi and ADP bound forms of MCAK for the microtubule lattice. Detyrosination also impairs microtubule disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that microtubule depolymerizing motors are directly inhibited by detyrosination of tubulin, resulting in stabilization of cellular microtubules. Detyrosination of transiently stabilized microtubules may give rise to persistent subpopulations of disassembly-resistant polymers to sustain sub-cellular cytoskeletal differentiation.
Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G 2 phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-Trityl-L-cysteine is a tight binding inhibitor (estimation of K i,app <150 nM at 300 mM NaCl and 600 nM at 25 mM KCl). S-Trityl-L-cysteine binds more tightly than monastrol because it has both an ϳ8-fold faster association rate and ϳ4-fold slower release rate (6.1 M ؊1 s ؊1 and 3.6 s ؊1 for S-trityl-L- Kinesins form a superfamily of motor proteins with about 14 different subfamilies clearly identified so far. They play important roles in intracellular transport and at different stages of cell division. The driving force behind these processes is ATP hydrolysis.The roles of different kinesins during cell division make them highly important for understanding fundamental aspects of mitosis and meiosis. In recent years, some of them have appeared as potential targets for anti-cancer drugs (1-3). One of these mitotic kinesins, human Eg5 (HsEg5/KSP), a member of the kinesin-5 family (4), is responsible for the formation and maintenance of the bipolar spindle (5). Eg5 represents an especially attractive target because when inhibited by microinjection with suitable antibodies (5), by RNAi 2 (6), or by treating cells with specific Eg5 inhibitors (7), it displays a very characteristic mitotic arrest phenotype, i.e. a monoastral spindle with an array of microtubules (MTs) emanating from a pair of nonseparated centrosomes surrounded by chromosomes. Cell-based as well as in vitro assays have led to the discovery of a series of inhibitors that target Eg5 and lead to mitotic arrest and cell death. Among these inhibitors are monastrol, the first Eg5 inhibitor discovered (7), terpendole E, identified from a fungal strain (8), HR22C16, structurally related to monastrol (9), CK0106023, a quinazolinone analogue representing the most potent Eg5 inhibitor identified so far (10), dihydropyrazoles (11), and S-trityl-L-cysteine (STLC) (12). Several of these inhibitors are currently intensively studied as potential anticancer drugs, as tools for studying fundamental processes in mitosis and function of its target (chemical genetics) (13), or simply as a model to understand the mechanisms of inhibition of this important class of proteins (14 -17).By using two small, preselected libraries from the NCI, we have recently identified several new inhibitors of human Eg5 activity, ...
When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 Å and compare it with a structure of the motor domain alone at 2.7 Å. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled-coil. This ‘double lockdown’, by cross-linking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release ADP. This autoinhibition mechanism could extend to some other kinesins.
The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in the assembly of the mitotic spindle. It shows a three-domain structure with an N-terminal motor domain, a central coiled coil, and a C-terminal tail domain. In vivo HsEg5 is reversibly inhibited by monastrol, a small cell-permeable molecule that causes cells to be arrested in mitosis. Both monomeric and dimeric Eg5 constructs have been examined in order to define the minimal monastrol binding domain on HsEg5. NMR relaxation experiments show that monastrol interacts with all of the Eg5 constructs used in this study. Enzymatic techniques indicate that monastrol partially inhibits Eg5 ATPase activity by binding directly to the motor domain. The binding is noncompetitive with respect to microtubules, indicating that monastrol does not interfere with the formation of the motor-MT complex. The binding is not competitive with respect to ATP. Both enzymology and in vivo assays show that the S enantiomer of monastrol is more active than the R enantiomer and racemic monastrol. Stopped-flow fluorometry indicates that monastrol inhibits ADP release by forming an Eg5-ADP-monastrol ternary complex. Monastrol reversibly inhibits the motility of human Eg5. Monastrol has no inhibitory effect on the following members of the kinesin superfamily: MC5 (Drosophila melanogaster Ncd), HK379 (H. sapiens conventional kinesin), DKH392 (D. melanogaster conventional kinesin), BimC1-428 (Aspergillus nidulans BimC), Klp15 (Caenorhabditis elegans C-terminal motor), or Nkin460GST (Neurospora crassa conventional kinesin).
Kinesin-12 (also called Kif15) is a mitotic motor protein that continues to be expressed in developing neurons. Depletion of kinesin-12 causes axons to grow faster, more than doubles the frequency of microtubule transport in both directions in the axon, prevents growth cones from turning properly, and enhances the invasion of microtubules into filopodia. These results are remarkably similar to those obtained in previous studies in which neurons were depleted of kinesin-5 (also called Eg5 or Kif11), another mitotic motor protein that continues to be expressed in developing neurons. However, there are also notable differences in the phenotypes obtained with depleting each of these motors. Depleting kinesin-12 decreases axonal branching and growth cone size, whereas inhibiting kinesin-5 increases these parameters. In addition, depleting kinesin-12 diminishes the appearance of growth-cone-like waves along the length of the axon, an effect not observed with depletion of kinesin-5. Finally, depletion of kinesin-12 abolishes the "waggling" behavior of microtubules that occurs as they assemble along actin bundles within filopodia, whereas inhibition of kinesin-5 does not. Interestingly, and perhaps relevant to these differences in phenotype, in biochemical studies, kinesin-12 coimmunoprecipitates with actin but kinesin-5 does not. Collectively, these findings support a scenario whereby kinesin-12 shares functions with kinesin-5 related to microtubule-microtubule interactions, but kinesin-12 has other functions not shared by kinesin-5 that are related to the ability of kinesin-12 to interact with actin.
Human Eg5, a mitotic motor of the kinesin superfamily, is involved in the formation and maintenance of the mitotic spindle. The recent discovery of small molecules that inhibit HsEg5 by binding to its catalytic motor domain leading to mitotic arrest has attracted more interest in Eg5 as a potential anticancer drug target. We have used hydrogen-deuterium exchange mass spectrometry and directed mutagenesis to identify the secondary structure elements that form the binding sites of new Eg5 inhibitors, in particular for S-trityl-l-cysteine, a potent inhibitor of Eg5 activity in vitro and in cell-based assays. The binding of this inhibitor modifies the deuterium incorporation rate of eight peptides that define two areas within the motor domain: Tyr125-Glu145 and Ile202-Leu227. Replacement of the Tyr125-Glu145 region with the equivalent region in the Neurospora crassa conventional kinesin heavy chain prevents the inhibition of the Eg5 ATPase activity by S-trityl-l-cysteine. We show here that S-trityl-l-cysteine and monastrol both bind to the same region on Eg5 by induced fit in a pocket formed by helix alpha3-strand beta5 and loop L5-helix alpha2, and both inhibitors trigger similar local conformational changes within the interaction site. It is likely that S-trityl-l-cysteine and monastrol inhibit HsEg5 by a similar mechanism. The common inhibitor binding region appears to represent a "hot spot" for HsEg5 that could be exploited for further inhibitor screening.
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