Copurification of kinesin polypeptides with microtubule-stimulated Mg-ATPase activity and kinetic analysis of enzymatic properties

Determination of kinetic properties for kinesin adenosine triphosphatase (ATPase), a proposed motor for transport of membranous organelles, requires adequate amounts of kinesin with a consistent level of enzymatic activity. A purification procedure is detailed that produces approximately 2 mg of kinesin at up to 96% purity from 800 g of bovine brain. This protocol consists of a microtubule affinity step using 5'-adenylylimidodiphosphate (AMP-PNP); followed by gel filtration, ion exchange, and hydroxylapatite chromatography; and then sucrose density gradient centrifugation. The microtubule-activated ATPase activity of kinesin coeluted with kinesin polypeptides throughout the purification. Highly purified kinesin had a Vmax of 0.31 mumol/min/mg in the presence of microtubules, with a Km for ATP of 0.20 mM. The kinetic constants obtained in these studies compare favorably with physiological levels of ATP and microtubules. Variations in buffer conditions for the assay were found to affect ATPase activity significantly. A study of the ability of kinesin to utilize a variety of cation-ATP complexes indicated that kinesin is a microtubule-stimulated Mg-ATPase, but kinesin is able to hydrolyze Ca-ATP, Mn-ATP, and Co-ATP as well as Mg-ATP in the presence of microtubules. In the absence of microtubules, Ca-ATP appears to be the best substrate. Studies with several inhibitors of ATPases determined that vanadate inhibited kinesin ATPase at the lowest concentrations of inhibitor, but significant inhibition of the ATPase also occurred with submillimolar concentrations of AMP-PNP. Other inhibitors of kinesin include N-ethylmaleimide, adenosine diphosphate (ADP), pyrophosphate, and tripolyphosphate. Further characterization of the kinetic properties of the kinesin ATPase is important for understanding the molecular mechanisms for transport of membranous organelles along microtubules.     

Mechanism of tail-mediated inhibition of kinesin activities studied using synthetic peptides

We used a truncated form of human conventional kinesin (K560) and a set of synthetic tail-derived peptides to investigate the mechanism by which the kinesin tail domain inhibits the protein's ATPase and motor activities. A peptide that spans residues 904-933 (C3) exhibited the strongest inhibitory effect on steady-state motility and ATPase activity. This inhibition reflected diminished binding of the ADP-bound kinesin head to the microtubule. Although peptide C3 bound to both K560 and microtubules, gliding assays using subtilisin-treated microtubules suggested that the binding to the microtubule contributes only little to the inhibition if there is sufficient affinity between the peptide and kinesin. We suggest that tail-mediated inhibition of kinesin activity is mainly the product of allosteric inhibition induced by the intramolecular binding of the kinesin tail domain to the motor domain, but simultaneous binding of the tail to the microtubule also may exert a minor effect.     

Direct inhibition of microtubule-based kinesin motility by local anesthetics

Local anesthetics are known to inhibit neuronal fast anterograde axoplasmic transport (FAAT) in a reversible and dose-dependent manner, but the precise mechanism has not been determined. FAAT is powered by kinesin superfamily proteins, which transport membranous organelles, vesicles, or protein complexes along microtubules. We investigated the direct effect of local anesthetics on kinesin, using both in vitro motility and single-molecule motility assays. In the modified in vitro motility assay, local anesthetics immediately and reversibly stopped the kinesin-based microtubule movement in an all-or-none fashion without lowering kinesin ATPase activity. QX-314, a permanently charged derivative of lidocaine, exerted an effect similar to that of lidocaine, suggesting that the effect of anesthetics is due to the charged form of the anesthetics. In the single-molecule motility assay, the local anesthetic tetracaine inhibited the motility of individual kinesin molecules in a dose-dependent manner. The concentrations of the anesthetics that inhibited the motility of kinesin correlated well with those blocking FAAT. We conclude that the charged form of local anesthetics directly and reversibly inhibits kinesin motility in a dose-dependent manner, and it is the major cause of the inhibition of FAAT by local anesthetics.     

Kinesin's tail domain is an inhibitory regulator of the motor domain

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.     

Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity

NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NOD(DTW) and NOD("DR2")) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport.     

Modification of the microtubule-binding and ATPase activities of kinesin by N-ethylmaleimide (NEM) suggests a role for sulfhydryls in fast axonal transport

N-Ethylmaleimide, an agent which alkylates free sulfhydryls in proteins, has been used to probe the role of sulfhydryls in kinesin, a motor protein for the movement of membrane-bounded organelles in fast axonal transport. When squid axoplasm is perfused with concentrations of NEM higher than 0.5 mM, organelle movements in both the anterograde and retrograde directions cease, and the vesicles remain attached to microtubules. Incubation of highly purified bovine brain kinesin with similar concentrations of NEM modifies the enzyme's microtubule-stimulated ATPase activity and promotes the binding of kinesin to microtubules in the presence of ATP. These results suggest that alkylation of sulfhydryls on kinesin alters the conformation of the protein in a manner that profoundly affects its interactions with ATP and microtubules. The NEM-sensitive sulfhydryls, therefore, may provide a valuable tool for the dissection of functional domains of the kinesin molecule and for understanding the mechanochemical cycle of this enzyme.     

ESR reveals the mobility of the neck linker in dimeric kinesin

Conventional kinesin is a highly processive motor that converts the chemical energy of ATP hydrolysis into the unidirectional motility along microtubules. The processivity is thought to depend on the coordination between ATPase cycles of two motor domains and their neck linkers. Here we have used site-directed spin labeling electron spin resonance (SDSL-ESR) to determine the conformation of the neck linker in kinesin dimer in the presence and absence of microtubules. The spectra show that the neck linkers co-exist in both docked and disordered conformations, which is consistent with the results of monomeric kinesin. In all nucleotide states, however, the neck linkers are well ordered when dimeric kinesin is bound to the microtubule. This result suggests that the orientation of each neck linker that is fixed rigidly controls the kinesin motion along microtubule tracks.     

Cotton GhKCH2, a plant-specific kinesin, is low-affinitive and nucleotide-independent as binding to microtubule

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, M396-734 corresponding to amino acids Q396-N734 was fused with 6xHis-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of M396-734 is not activated by Ca2+, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 microM, much higher than that of other kinesins reported. The cosedimentation assay indicated that M396-734 could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.     

Genetic engineering of a Ca(2+) dependent chemical switch into the linear biomotor kinesin

Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca(2+)-calmodulin, the association of the engineered kinesin with microtubules showed a steep Ca(2+)-dependency in ATPase activity at Ca(2+) concentrations of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca(2+)-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca(2+)-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.     

Kinesin is involved in protecting nascent microtubules from disassembly after recovery from nocodazole treatment

Upon recovery from nocodazole treatment, microtubules from cultured epithelial cells exhibit unusual properties: they re-grow as fast as any highly dynamic microtubule, but they are also protected against disassembly when challenged with nocodazole like the stable microtubules of steady-state cells. Exploring the mechanism that underlies this protection, we found that it was sensitive to ATP treatment and that it involved conventional kinesin. Kinesin localized at the growing end or along nascent microtubules. Its inhibition using a dominant-negative construct for cargo binding, or by micro-injecting an anti-kinesin heavy chain antibody that impairs motor activity, resulted in the partial or total loss of microtubule protection. Finally, in an ex vivo elongation assay, we found that kinesin also participates in the control of microtubule re-growth. Altogether, our findings suggest that kinesin is involved in an early microtubule protection process that is linked to the control of their dynamics during their early growth phase.     

The kinesin family member BimC contains a second microtubule binding region attached to the N terminus of the motor domain

The kinesin family member BimC has a highly positively charged domain of approximately 70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by approximately 7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mm potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in kcat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity.     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.     

Microtubule-associated proteins and microtubule-based translocators have different binding sites on tubulin molecule

It has been previously shown that a class of microtubule proteins, the so-called microtubule-associated proteins (MAPs), binds to the C-terminal part of tubulin subunits. We show here that microtubules composed of tubulin whose 4-kDa C-terminal domain was cleaved by subtilisin (S-microtubules) are unable to bind MAPs but can still bind the anterograde translocator protein kinesin and the retrograde translocator dynein. Binding of both motors to S-microtubules, like their binding to normal microtubules, was ATP-dependent. In addition, direct competition experiments showed that binding sites for kiensin and MAPs on the microtubule surface lattice do not overlap. Furthermore, S-microtubules stimulated the ATPase activity of kinesin at least 8-fold, and the affinities of kinesin for control and S-microtubules were identical. S-microtubules were able to glide along kinesin-coated coverslips at a rate of 0.2 microns/s, the same rate as control microtubules. We conclude, that unlike MAPs, kinesin and cytoplasmic dynein bind to the tubulin molecule outside the C-terminal region.     

45 kDa fragment of the kinesin molecule possesses high ATPase activity and binds to microtubules

Kinesin is a mechano-chemical ATPase capable to move particles along microtubules and microtubules along the solid substrate. Molecule of bovine brain kinesin is a heterotetrameric unit consisting of two heavy (120 kDa) and two light (62 kDa) chains. We used limited proteolysis to study the location of the functional sites on the kinesin molecule. Chymotrypsin cleavage produced a stable 45 kDa fragment of the heavy chain which was purified from the digest using FPLC chromatography on a Superose 12 column. 45 kDa fragment contained both a microtubule-binding site and a ATPase site of the kinesin molecule. Cleavage of the 45 kDa fragment from the rest of the heavy chain significantly activated its ATPase activity. However, this activity remained fully dependent on microtubules. We suggest that the chymotrypsin cleavage uncouple ATPase activity of kinesin (found in the 45 kDa fragment) from its translocator activity (which, probably, required the presence of other parts of the molecule).     

Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans

C. elegans contains a microtubule binding protein that resembles both dynein and kinesin. This protein has a MgATPase activity and copurifies on both sucrose gradients and DEAE Sephadex columns with a polypeptide of Mr approximately 400 kd. The ATPase activity is 50% inhibited by 10 microM vanadate, 1 mM N-ethyl maleimide, or 5 mM AMP-PNP; it is enhanced 50% by 0.2% Triton. The 400 kd polypeptide is cleaved at a single site by ultraviolet light in the presence of ATP and vanadate. In these ways, the protein resembles dynein. The protein also promotes ATP-dependent translocation of microtubules or axonemes, "plus" ends trailing. This property is kinesin-like; however, the motility is blocked by 5 microM vanadate, 1 mM N-ethyl maleimide, 0.5 mM ATP-gamma-S, or by ATP-vanadate-UV cleavage of the 400 kd polypeptide, characteristics that differ from kinesin. We propose that this protein is a novel microtubule translocator.     

Evidence that the 116 kDa component of kinesin binds and hydrolyzes ATP

Kinesin was prepared from bovine brain as described previously for studies of translocation. A major component of kinesin, (116 kDa) was shown to undergo specific photocrosslinking with [alpha-32P]ATP, indicating it was an ATP-binding polypeptide. A low ATPase activity associated with kinesin was stimulated up to 5-fold by microtubules to a specific activity of 14 nmol . min-1 . mg-1. N-Ethylmaleimide inhibited both [alpha-32P]ATP binding to the 116 kDa polypeptide and microtubule-stimulated ATPase activity, suggesting that the 116 kDa polypeptide was the catalytic subunit of kinesin. Though the ATPase activity associated with kinesin is low, it may be sufficient to support motility assuming it is coupled to the velocity of translocation.     

Insight into the molecular mechanism of the multitasking kinesin‐8 motor

Members of the kinesin‐8 motor class have the remarkable ability to both walk towards microtubule plus‐ends and depolymerise these ends on arrival, thereby regulating microtubule length. To analyse how kinesin‐8 multitasks, we studied the structure and function of the kinesin‐8 motor domain. We determined the first crystal structure of a kinesin‐8 and used cryo‐electron microscopy to calculate the structure of the microtubule‐bound motor. Microtubule‐bound kinesin‐8 reveals a new conformation compared with the crystal structure, including a bent conformation of the α4 relay helix and ordering of functionally important loops. The kinesin‐8 motor domain does not depolymerise stabilised microtubules with ATP but does form tubulin rings in the presence of a non‐hydrolysable ATP analogue. This shows that, by collaborating, kinesin‐8 motor domain molecules can release tubulin from microtubules, and that they have a similar mechanical effect on microtubule ends as kinesin‐13, which enables depolymerisation. Our data reveal aspects of the molecular mechanism of kinesin‐8 motors that contribute to their unique dual motile and depolymerising functions, which are adapted to control microtubule length.     

Loading direction regulates the affinity of ADP for kinesin

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.     

The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.     

Inhibitors of kinesin activity from structure-based computer screening

Kinesin motor proteins use ATP hydrolysis for transport along microtubules in the cell. We sought to identify small organic ligands to inhibit kinesin's activity. Candidate molecules were identified by computational docking of commercially available compounds using the computer program DOCK. Compounds were docked at either of two sites, and a selection was tested for inhibition of microtubule-stimulated ATPase activity. Twenty-two submillimolar inhibitors were identified. Several inhibitors appeared to be competitive for microtubule binding and not for ATP binding, and three compounds showed 50% inhibition down to single-digit micromolar levels. Most inhibitors grouped into four distinct classes (fluoresceins, phenolphthaleins, anthraquinones, and naphthylene sulfonates). We measured the binding of one inhibitor, rose bengal lactone (RBL), to kinesin (dissociation constant 2.5 microM) by its increase in steady-state fluorescence anisotropy. The RBL binding site on kinesin was localized by fluorescent resonance energy transfer (FRET) using a donor fluorophore (coumarin) covalently attached at unique, surface-exposed cysteine residues engineered at positions 28, 149, 103, 220, or 330. RBL was found to bind in its original docked site: the pocket cradled by loop 8 and beta-strand 5 in kinesin's three-dimensional structure. These results confirm this region's role in microtubule binding and identify this pocket as a novel binding site for kinesin inhibition.