(Received for publication, July 25, 1996, and in revised form, December 16, 1996)
From the Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213
Motor domains of kinesin were expressed that
extend from the N terminus to positions 346, 357, 365, 381, and 405 (designated DKH346-DKH405) to determine if the kinetic differences
observed between monomeric DKH340 and dimeric DKH392 (Hackney, D. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6865-6869)
were specific to these constructs or due to their oligomeric state.
Sedimentation analysis indicated that DKH346, DKH357, and
DKH365 are predominantly monomeric and that DKH381 and DKH405 are
predominantly dimeric at 0.01-0.03 µM, the
concentrations used for ATPase assays. In buffer with 25 mM
KCl, all have high kcat values of 38-96
s1 at saturating microtubule (MT) levels. Monomeric
DKH346, DKH357, and DKH365 have K0.5(MT) values
of 17, 9, and 1.4 µM, respectively, but the
K0.5(MT) values for the dimeric species are
significantly lower, with 0.02 and 0.14 µM for DKH381 and
DKH405, respectively. The three new monomers release all of their ADP
on association with microtubules, whereas the two new dimers retain
approximately half of their ADP, consistent with the half-site
reactivity observed previously with dimeric DKH392. Both the
kbi(ATPase)
(=kcat/K0.5(MT)) values
for stimulation of ATPase by MTs and the
kbi(ADP) for stimulation of ADP release by MTs
were determined in buffer containing 120 mM potassium
acetate. The ratio of these rate constants
(kbi(ratio) = kbi(ATPase)/kbi(ADP))
is 60-100 for the dimers, indicating hydrolysis of many ATP molecules
per productive encounter with a MT as observed previously for DKH392
(Hackney, D. D. (1995) Nature 377, 448-450). For the
monomers, kbi(ratio) values of ~4 indicate
that they also may hydrolyze more than one ATP molecule per encounter
with a MT and that the mechanism of hydrolysis is therefore
fundamentally different from that of actomyosin. DKH340 is an exception
to this pattern and may undergo uncoupled ATP hydrolysis.
Kinesin is a molecular motor that is capable of producing movement along microtubules (see Refs. 1-3 for review). A striking feature of its motility is that a single molecule of native dimeric kinesin is able to attach to a microtubule (MT)1 and slide along it without net dissociation (4-8). This is in contrast to the movement of myosin along actin, which is believed to be nonprocessive.
The N-terminal 340 amino acids of kinesin are conserved among all
superfamily members and contain the sites for ATP hydrolysis and
interaction with MTs. The structure of this domain has recently been
solved by x-ray crystallography (9). The region between amino acids 340 and 400 is predicted to be -helical and is likely to contain a
region of coiled-coil (10, 11). Previous work has established that
DKH340 (containing amino acids 1-340) is monomeric, with an
s20,w value of 3.3 S, whereas DKH392 (containing amino acids 1-392) is dimeric, with an
s20,w value of 5.2 S (11). This led to a
revised domain model of kinesin in which the two minimal motor domains
are dimerized through interaction of the coiled-coil region in the neck
without a highly flexible hinge at their point of attachment to the
neck (11). The poorly conserved region around position 400 is not
predicted to readily form a coiled-coil structure, and it likely serves
as a flexible hinge that attaches a functional dimer head unit to the
rest of the coiled-coil stalk (12). Subsequent determination of the oligomeric state of constructs of different lengths and from other species has confirmed this pattern and refined the region required for
dimerization (7, 8, 13, 14). A high degree of processive behavior
likely requires the coordination of two head domains in a dimer as only
dimeric constructs of kinesin can track in a linear path down a MT
(7).
The various head constructs of kinesin have a wide range of reported ATPase properties (7, 8, 10, 15-19). This reflects both the intrinsic differences between constructs due to their length, oligomeric state, and species of origin and differences in assay conditions, particularly the ionic strength and the concentration of heads. The work presented here is an examination of seven head constructs of Drosophila kinesin that differ in the amount of the neck domain that is included. This extensive series of constructs allows systematic evaluation of the influence of the length of the neck on the oligomeric state as well as evaluation of the influence of dimerization on kinetic properties under uniform assay conditions. These constructs exhibit a large range of properties, which suggests that caution must be exercised to prevent overinterpretation of results with a more limited series. Despite this variation, however, several characteristic differences between dimers and monomers have emerged.
The plasmids pDKH346, pDKH357, pDKH365, pDKH381, and pDKH405 were obtained by polymerase chain reaction with the proofreading Pfu polymerase essentially as described for pDKH340 and pDKH392 (11, 20). Given the wide range of kinetic properties observed here with head constructs that all contain the same catalytic domain, it is reasonable to be concerned that differences may result from spurious mutations that were introduced in some constructs during cloning. All of the constructs used here were obtained by subcloning into the same pDKH392 plasmid, and thus, all have the same N-terminal region from amino acids 1 to 295. Only the very C-terminal part (from the PstI site corresponding to amino acid 295) was derived by polymerase chain reaction, and the DNA sequence of the polymerase chain reaction-derived region was determined and shown not to contain any errors. DNA sequences were obtained by the DNA Sequencing Facility of the University of Pittsburgh by the dye terminator method. As a further check for possible introduction of mutations into the N-terminal region during subcloning, the PstI tail fragment of the clone of pDKH357 that was used for most of the protein preparations was excised and religated into a pDKH392 backbone. DKH357 protein expressed by this new construct had the same high K0.5(MT) value as the original DKH357 clone, indicating that the difference in K0.5(MT) between DKH357 and DKH392 is not due to unintended changes in sequence in the catalytic region.
Preparations of the head domain proteins used in this work (including
new preparations of DKH392 and DKH340) were performed essentially as
described previously for DKH340 and DKH392 (11, 20), except that the
exposure to excess EDTA during lysis and sonification was eliminated.
Final preparations were stored in 50% glycerol at 80 °C.
All reactions were performed at 25 °C in A25 buffer as described
previously (20), except that 0.1 mg/ml bovine serum albumin was not
routinely included. For ATPase reaction, A25 buffer was supplemented
with 1 mM MgATP, 2 mM P-enolpyruvate, 0.3 mM NADH, and pyruvate kinase and lactate dehydrogenase at
3-5 µg/ml. Reactions were also supplemented with KCl or potassium
acetate as indicated, and solutions containing MTs were supplemented
with 3-10 µM Taxol. ATPase reactions were initiated by
addition of a small volume of an intermediate stock of head domain at
typically 0.5-2 µM in buffer supplemented with 0.1 mM ATP. The protein concentration was determined by the
Bradford method (36) on this intermediate stock using the Coomassie
Plus kit (Pierce) with bovine serum albumin as standard. ATPase rates
are reported on a per site (head) basis whether the construct is
monomeric or dimeric. kcat and K0.5(MT) values were determined in triplicate
and are reported as ±S.E. The rate of ADP release from the head domain
was determined using [-32P]ADP and a cold chase with
excess ATP as described (20). The first-order rate constants for ADP
release were obtained by linear regression on a plot of ln([bound
ADP]) versus time for typically five points between 10 and 90% reaction. The standard error of the fit was
5%.
kbi(ADP) values were determined from the
increase in ADP release rate on addition of MTs and are reported as the average of at least two determinations at different MT concentrations.
Tubulin was isolated by two cycles of polymerization and chromatography
on phosphocellulose (21) and polymerized with Taxol in A25 buffer with
25 mM KCl as described (20). In some cases, the MTs were
further concentrated, and unpolymerized tubulin was removed by
centrifugation at 22 °C (30 min at 150,000 × g) and gentle resuspension with a loose-fitting Teflon pestle at room temperature in A25 buffer with 25 mM KCl and 10 µM Taxol. Following resuspension, the MTs were
centrifuged for 1.5 min at 16,000 × g to remove
aggregated material. MT stock solutions were divided into small
aliquots, frozen rapidly in liquid nitrogen, and stored at 80 °C.
For use in experiments, a tube of polymerized MTs was rapidly thawed by
incubation in a water bath at 25 °C. One freeze-thaw cycle produced
no detectable change in the ability of MTs to stimulate the ATPase of
head constructs. MT concentrations are reported as the concentration of
tubulin
-heterodimers.
Hydrodynamic characterization was performed essentially as described previously (11). Sucrose gradient centrifugation was performed for 19 h at 4 °C in A25 buffer with 25 mM KCl and 0.1 mM MgATP at 41,000 rpm in either an SW 41 (Beckman Instruments) or a TH641 (DuPont) rotor. In some cases, gradients also contained NaCl or 120 mM potassium acetate (without KCl) as indicated. Catalase, bovine serum albumin, and carbonic anhydrase at 0.1-0.2 mg/ml were routinely included as standards. Gradients were fractionated into 20 drop fractions (typically 14 full fractions) and analyzed by SDS-polyacrylamide gel electrophoresis with staining by Coomassie Blue. The s20,w value of the kinesin head was determined using the least-squares fit of the s20,w values of the standards versus their migration. Gel filtration on Sephacryl S-300 (Pharmacia Biotech Inc.) was conducted at ~22 °C in A25 buffer with 25 mM KCl, 0.1 mM MgATP, and bovine serum albumin as an internal marker as described (11). Fractions from centrifugation or chromatography were analyzed by SDS-polyacrylamide gel electrophoresis with staining by Coomassie Blue. For experiments at very low concentration of head domain, the head domain was concentrated from the bulk of each gradient fraction by adsorption to phosphocellulose and elution with SDS sample buffer and then analyzed by polyacrylamide gel electrophoresis with detection by Western blot analysis using SUK4 (22) as the primary antibody as described (23).
Previous work (11) has
established that DKH340 is monomeric, with an
s20,w value of 3.3 S, whereas DKH392 is
dimeric, with an s20,w value of 5.2 S,
and further work (7, 8, 13, 14) has confirmed and extended this
conclusion with other constructs of similar length. To better localize
the regions required for dimerization and to determine the influence of
dimerization itself, kinesin head domains terminating at a series of
positions in the neck region were expressed in Escherichia
coli and purified essentially as described previously (11, 20).
The velocity of sedimentation during centrifugation in a sucrose
gradient was determined over a range of head concentrations in A25
buffer with 25 mM KCl, and the
s20,w values are summarized in Table I. At a concentration of 1 µM, DKH346,
DKH357, and DKH365 have s20,w values of
3.5-3.6 S, which are similar to the values for DKH340, which was
previously shown to be monomeric (11), whereas DKH381 and DKH405 have
s20,w values of 5.1-5.7 S, which are
similar to the 5.2 S value for dimeric DKH392 (11). Gel filtration
experiments on Sephacryl S-300 (data not shown) indicate that DKH346,
DKH357, and DKH365 elute at the same position as DKH340 and thus have
similar diffusion coefficients, whereas DKH381 and DKH405 elute at the
same position as DKH392. The similarity of both the sedimentation and
diffusion coefficients of DKH346, DKH357, and DKH365 to monomeric
DKH340 and of DKH381 and DKH405 to dimeric DKH392 indicates that these
constructs are also predominantly monomeric and dimeric, respectively,
under these conditions as summarized in Table II.
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Sedimentation of DKH381-DKH405 at a higher initial head concentration
of 20 µM results in increased
s20,w values (Table I), indicating
aggregation to species larger than the dimer. Aggregation is
particularly severe for DKH381 and is highly dependent on the salt
concentration as indicated in Fig. 1. When 100 or 300 mM NaCl was included, DKH381 sedimented with
s20,w values of 5.1 and 5.0 S,
respectively (Fig. 1, A and B). An
s20,w value of 5.0 S was also obtained
with 1000 mM NaCl (data not shown). These
s20,w values are consistent with no
aggregation beyond the dimer at higher ionic strength. With no added
NaCl and only 25 mM KCl, DKH381 migrates more rapidly at
~8.0 S (Fig. 1C). There is little DKH381 remaining in the
position expected for the dimer, and the peak is unsymmetrical, with
streaking toward the dimer position. This behavior is consistent with
an equilibrium between dimers and higher aggregates that favors
aggregates at this high concentration, but is reversible on the time
scale of sedimentation. In the complete absence of added salt, DKH381
is largely insoluble at 20 µM and has a visible
turbidness that is cleared by brief centrifugation at 13,000 × g with removal of the most of the protein. DKH381 remaining
in solution sediments at ~9 S (Fig. 1D). Aggregation of
DKH381 is also reduced by 120 mM potassium acetate (Fig.
1E), although the value of 5.7 S indicates that dissociation
to dimers is not complete. The value of 5.2 S for DKH392 and DKH405 in
120 mM potassium acetate (Table I) indicates that these
constructs are not aggregated even at an initial loading of 20 µM. A more detailed analysis of the oligomeric state
during ATPase reactions is given below following description of the
ATPase properties of the different constructs.
Stoichiometry of ADP Release on Binding to MTs
The new head
constructs contain tightly bound ADP that can be labeled by incubation
with [-32P]ATP as described previously for native
kinesin (24), DKH340 (20), and DKH392 (11). Monomeric DKH340 releases
essentially all of its bound ADP on binding to MTs, while dimeric
DKH392 releases only half of its ADP (25), and this half-site
reactivity of dimeric species is likely involved in generation of the
high degree of processivity of kinesin. Analysis of the other head
constructs (Table II) indicates that all of the monomeric constructs
release ~95% of their ADP on binding to MTs, while all of the
dimeric constructs release ~60% of their ADP after 3 s. This
release is consistent with the time course reported for DKH340 and
DKH392 (25) and indicates that half-site reactivity is restricted to dimeric species.
The rate of release of the bound
[-32P]ADP during a chase with excess unlabeled ATP in
the absence of MTs (ko) equals the steady-state
ATPase rate for both bovine kinesin (24, 26) and isolated head domain
constructs (20, 27, 28). For monomeric DKH340, ko
had previously been shown to be 5-fold higher than the rate of ADP
release from dimeric DKH392 (0.025 versus 0.005 s
1) (11, 20) in 25 mM KCl. Analysis of the
ADP release rates for all seven head constructs in 25 mM
KCl (Table II) indicates that this difference is not due to
dimerization because all the other heads, both monomers and dimers,
have low ADP release rates of 0.005-0.010 s
1. The higher
rate with DKH340 rather represents an abnormally high basal ADP release
rate for DKH340. A similar pattern is observed in 120 mM
potassium acetate (Table III), with all the rates
~2-fold higher.
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The MT-stimulated ATPase kinetics of the
heads in 25 mM KCl were determined as indicated in Table
II. All of the heads have high kcat values, but
monomers have higher values of 61-96 s1 compared with
dimers at ~40 s
1. In contrast to the relatively similar
kcat values, the K0.5(MT) values differ dramatically over almost a 1000-fold range (from 0.02 µM for dimeric DKH381 to 17 µM for
monomeric DKH346). All three dimeric constructs have extremely low
K0.5(MT) values of
0.14 µM when
assayed at ~0.01 µM head concentration, whereas the
monomeric constructs have much higher K0.5(MT)
values of 1.4-17 µM, with the exception of DKH340. The
results of analysis in 120 mM potassium acetate are
indicated in Table III, and the pattern is similar, but the
K0.5(MT) values are higher due to the higher ionic strength. The K0.5(MT) values for the
monomers were not determined because they would be too high to measure
in most cases, but the bimolecular rate constant
(kbi(ATPase)) was still determined from the MT
dependence of the ATPase rate at low MT concentrations. The stimulation
of the ATPase by MTs exhibits approximately hyperbolic saturation
behavior under these conditions, and thus,
kbi(ATPase) determined in this way is
numerically equivalent to
kcat/K0.5(MT).
The kcat for dimeric DKH392 has been shown to be
independent of the concentration of DKH392 in the assay over the range
of 0.004-0.025 µM (23) in A25 buffer with 25 mM KCl. This is the maximum range over which the coupled
assay using pyruvate kinase and lactate dehydrogenase is readily
applicable. This range was extended to 0.5 µM DKH392 in
the absence of pyruvate kinase by determining the initial rate of
Pi production using the malachite green method (29). Over
the range of 2.5-25 µM MTs, the ATPase rate with 0.5 µM DKH392 varied between 36 and 42 s1, with
an extrapolated kcat of 41 s
1.
Thus, there is no significant change in the kcat
when assayed at high concentrations of dimeric DKH392.
The oligomeric state of the constructs during an ATPase assay is influenced by the way in which the assay is conducted. Typically, the assay is initiated by dilution of an intermediate stock of head domain at 0.5-2 µM into the reaction mixture with spectrophotometric determination of the initial rate over 30-60 s. The final concentration of head domain is typically 0.01-0.03 µM and was ~0.01 µM for Tables II and III. The results of Table I indicate that DKH340-DKH365 have little tendency to aggregate even at 20 µM, which is ~1000-fold greater than the concentration in the ATPase assay. Thus, DKH340-DKH365 are predominantly monomeric under the conditions of the ATPase assay. Table I also indicates that DKH381, DKH392, and DKH405 are predominantly dimeric in 120 mM potassium acetate, even at high concentration, and thus will not be aggregated at the lower concentrations of the ATPase assay. The dimers will also not be aggregated at ~0.01 µM in ATPase reactions with 25 mM KCl, but some further aggregation may be present in the intermediate stock solution of 0.5-2 µM, particularly for DKH381.
Several lines of evidence indicate that higher aggregates of
DKH381-DKH405 dissociate rapidly to dimers on dilution into the ATPase
assay and that the observed ATPase kinetics are due predominantly to
dimeric species. If the higher aggregates dissociate slowly and have
different kinetics from the dimeric species, then the ATPase properties
of aggregates (fraction 6 in Fig. 1C) should differ from
those of unaggregated dimers (fraction 10 in Fig. 1A). One
potential consequence of dissociation of aggregates over the course of
an ATPase reaction is that a lag or a burst would be observed on
dilution of fraction 6 (Fig. 1C) into the ATPase assay. The
results of Fig. 2, however, indicate that the ATPase reaction of fraction 6 is highly linear. Any lag or burst is either completed within the few seconds required for mixing and response of
the coupled assay system or is longer than the 200 s for which the
reaction remains approximately linear. The possibility that the linear
ATPase kinetics of fraction 6 are due to aggregates that do not
dissociate over the ~200 s of total reaction is made unlikely by the
fact that the unaggregated fraction 10 of the gradient with 300 mM NaCl (Fig. 1A) exhibits essentially identical linear ATPase kinetics when diluted to the same final concentration (data not shown). Furthermore, analysis of the ATPase of these two
gradient fractions over a range of MT concentrations indicates that
they have essentially the same kcat and
K0.5(MT) values (44.7 ± 0.7 and 31.5 ± 1.8 s
1 and 0.032 ± 2 and 0.032 ± 2 µM for fraction 6 (Fig. 1C) and fraction 10 (Fig. 1A), respectively). The lower apparent
kcat for fraction 10 of the gradient with 300 mM NaCl is expected due to the presence of bovine serum
albumin in this fraction (Fig. 1A). These
kcat and K0.5(MT) values
are similar to those reported in Table II for DKH381 with consideration
of the different head concentrations during the assay and different
batches of MTs. The similarity of these kinetic properties indicates
either that any aggregates present in fraction 6 of the gradient
without added NaCl dissociate rapidly to dimers following dilution into
the ATPase assay or that the aggregates have the same kinetic
properties as unaggregated DKH381 dimers present in fraction 10 of the
gradient with 300 mM NaCl. Similar analysis of the peak
fractions of the gradients at 20 µM for DKH381 with 100 mM NaCl or with 120 mM potassium acetate and
for DKH392 and DKH405 without added NaCl or with 120 mM
potassium acetate indicated that there were no significant differences
in kcat or K0.5(MT)
values from the results of Tables II and III.
Conversely, there is no evidence for significant dissociation of
DKH381-DKH405 to monomers at higher dilution in the ATPase reaction.
If dissociation to monomer occurs during the time course of an ATPase
reaction and if the rates for the dimer and monomer differ at that MT
concentration, then a nonlinear ATPase progress curve would be
produced. In particular, the large increase in K0.5(MT) for monomers versus dimers
should produce a significant decrease in ATPase rate at low MT
concentration on dissociation of a dimer to a monomer. Dimers at low MT
levels do not, however, show a major loss of activity with time at high
dilution during an ATPase reaction. When a reaction at 0.0056 µM DKH381 and 0.01 µM MTs is followed over
a long time, the rate after 1200 s is still 73% of the initial
rate. Similar small decreases in ATPase rate with time are observed
with monomeric constructs and are possibly due to slow denaturation or
adsorption to the cuvette. If the monomer of DKH381 had the same
kinetic properties as monomeric DKH365, then the rate would have
decreased on dissociation of DKH381 to monomers from the observed
initial value of 16 s1 to 0.48 s
1 based on
the kcat and K0.5(MT)
data of Table II. As only a minor decrease in rate of 27% was
observed, it is likely that there is no extensive dissociation to
monomer over 1200 s and that the initial rate corresponds
exclusively to that of the starting dimeric species.
The rate of MT stimulation of
release of [-32P]ADP (kbi(ADP))
was determined in 120 mM potassium acetate for comparison
with the kbi(ATPase) values as indicated in
Table III. The maximum kbi(ADP) value occurs
with DKH381, and progressively decreasing values are observed for both
longer and shorter constructs, with the exception of DKH340. For
both monomers and dimers, the changes in
kbi(ADP) parallel the changes in
kbi(ATPase), but at different absolute values.
Thus, the kbi(ratio) of
kbi(ATPase)/kbi(ADP) is
~4 for the three monomers DKH346, DKH357, and DKH365 despite considerable variation in the individual values of
kbi(ATPase) and kbi(ADP).
For the dimers DKH381, DKH392, and DKH405, the
kbi(ratio) values are also similar to each
other, but at a higher value of 60-100, while the
kbi(ATPase) values vary by >6-fold.
Secondary structure predictions indicate that residues 340 to
~390 of Drosophila kinesin are highly conserved and are
likely to be -helical and to form a coiled-coil neck that connects
the globular head (motor) domain of residues 1-340 to the hinge in the
region of proline 399 (see Figs. 5 and 7 of Ref. 11). DKH340 lacks the
neck region between amino acids 340 and 390 and is monomeric, whereas
DKH392 is dimeric. Thus, the neck region between amino acids 340 and
392 is necessary for dimerization through what is likely a coiled-coil
interaction. The more refined deletion analysis presented here
indicates that constructs up to the size of DKH365 remain monomeric,
but DKH381 and larger constructs are dimers that remain associated even
at the low concentrations used in ATPase assays. The region between
amino acids 346 and 365 is highly charged, and this may contribute to
its inability to associate tightly enough to effect dimerization by
itself. Of the 20 residues in this region, 5 are negatively charged,
and 9 are positively charged. In addition, there is a gap in the
hydrophobic heptad repeat in this region with a d position
occupied by Glu-355 and an a position occupied by Asn-359.
The region between amino acids 362 and 376 contains hydrophobic
residues in the heptad repeat positions, and the requirement of this
region for stable dimerization suggests that this hydrophobic
interaction makes a major contribution to dimerization.
These results are in general agreement with recent work on other related constructs (7, 8, 13, 14). In particular, Correia et al. (13) reported that similar constructs of Drosophila kinesin designated K341 and K366 were monomeric and that K401 was dimeric. This work corrects an early report that K401 was monomeric by scanning electron microscopy (27). The dimeric constructs, particularly DKH381, exhibit an increase in the apparent s20,w value at high concentration, and this is likely due to formation of higher aggregates. Similar aggregation was reported for K401 (13). Under the conditions of the ATPase assay, however, these aggregates are likely dissociated to dimers as indicated by ATPase analysis of aggregated DKH381 in 25 mM KCl (Fig. 2) and by the lack of extensive aggregation in 120 mM potassium acetate even at 20 µM DKH381. Some analogous dimeric constructs have been reported to dissociate to monomers at the low concentrations typical of ATPase assays (13, 30). Analysis of DKH381-DKH405 indicates that they remain dimeric at the concentrations at which we performed ATPase reactions and that there is no indication of dissociation at long times. The estimation of dissociation of the dimers to monomers at very low concentration is complicated by a number of technical factors, particularly the tendency for these head constructs to adsorb to surfaces. This problem prevented our application of gel filtration chromatography to analysis of DKH381. Adsorption of K401 to the walls of the cuvette during equilibrium centrifugation was also extensive and prevented analysis by Correia et al. (13) at low concentrations. Their value of 0.037 µM for dissociation of K401 was therefore based on experiments at high initial concentrations (0.5-3 µM), and even this estimation still required a large correction for adsorption. Gradient centrifugation in large diameter tubes is not significantly affected by adsorption, and this method indicates that DKH381 remains predominantly dimerized even at an initial concentration of 0.02 µM. The lack of any significant dissociation of DKH381, DKH392, and DKH405 during the initial rate phase of an ATPase assay is further supported by the gel filtration results with DKH405 at low concentration, the relative linearity observed in the ATPase reaction of DKH381 even at high dilution, and the invariance in kcat for DKH392 over a range of 0.004-0.5 µM.
The monomeric constructs reported here have higher
kcat values than the dimeric constructs, but
this difference is comparatively modest (61-64 versus
38-44 s1), with the exception of DKH340. This decreased
kcat for dimers relative to monomers is
consistent with only one head of a dimer being active at a time due to
the half-site reactivity. Other work (30) has indicated that a fusion
protein of human kinesin analogous to K401 is dimeric, but with a high
Kd value of 0.7 µM and thus is mainly
dissociated at low concentration. Analysis of the ATPase properties of
this construct has led to the proposal (30) that dimers should have
significantly lower kcat values of ~10
s
1 compared with monomers with
kcat values of ~55 s
1. Given the
extreme variation in the kinetic properties with small changes in
length of the neck region, even when the sequence is still wild-type,
the influence of a non-wild-type extension in this case is hard to
predict. Conclusions based on fusion proteins in this sensitive area
may not be applicable to any native constructs. The work presented here
establishes that high kcat values of ~40 s
1 are characteristic of native Drosophila
dimeric constructs. The much lower kcat values
observed with some other preparations are likely due to factors besides
dimerization itself. An additional consideration is that active dimers
must have kcat values of ~40 s
1
to produce the velocity of sliding that is observed in single motor
motility as discussed previously (25).
That the seven head constructs investigated here should all exhibit low basal rates and high extents of stimulation by MTs is not surprising given that they all have the same catalytic domain. What is surprising is the wide variation in the effectiveness of MTs for producing this stimulation. For example, the addition of only 6 amino acids between DKH340 and DKH346 results in an almost 100-fold change in K0.5(MT) values (0.176 versus 16.8 µM). Equally striking is the extremely low K0.5(MT) value of 0.02 µM for DKH381. This K0.5(MT) value is similar in magnitude to the 0.011 µM concentration of DKH381 that was used in the ATPase measurements. Consequently, even this low K0.5(MT) value must be considered an overestimate because the free MT level will be less than the total MT level due to depletion of free MTs by binding to DKH381 in this tight binding situation. Thus, the K0.5(MT) values span a range of ~1000-fold (from <0.02 to 16.8 µM). Clearly, conclusions based on a single construct, or even a limited number of constructs, could give results that may not be characteristic of constructs of even very similar size. For example, which of the constructs DKH340-DKH365 should be used as a "generic" monomer for purposes of comparison with the dimeric constructs? Even within the three dimers, the K0.5(MT) values differ by 7-fold.
What then are the defining kinetic characteristics that differentiate monomer heads from dimers of heads? Tables II and III indicate that there are two significant discontinuities in kinetic properties that occur at the monomer-dimer transition. One clear difference is that all three dimeric constructs release half of their ADP on binding to a MT, whereas all four monomers release essentially all of their ADP (Table II). Thus, half-site interaction with MTs is a characteristic property of native dimeric head species and not just a property of DKH392 (25). The other conspicuous difference is that dimers have kbi(ratio) values that are >50, whereas monomers have low kbi(ratio) values of ~4, with the exception of DKH340 (Table III). The analysis is complicated by the fact that DKH340 is very different from the other monomer heads. As discussed below, this possibly reflects uncoupled hydrolysis by DKH340, and it will be considered separately.
The high kbi(ratio) values for the dimers are consistent with highly processive movement along MTs in which many ATP molecules are hydrolyzed during each productive encounter of a dimer with a MT, as developed previously (31). In this analysis, kbi(ADP) is the net bimolecular rate for a head-ADP complex to bind to a MT with productive release of ADP, and kbi(ATPase) is the net bimolecular rate for stimulation of ATP hydrolysis by interaction with a MT. The ratio of these rates constants (kbi(ratio)) is the average number of ATP molecules hydrolyzed per productive encounter of a head-ADP complex with a MT. The kbi(ratio) value of ~4 for the monomers is notable for both the fact that it is much less than the value of >50 for the dimers and for the fact that it is still significantly greater than 1. The large decrease in kbi(ratio) for the monomers indicates that they should not exhibit extensive processivity of movement because they can take no more than four steps on average before diffusional separation from the MT. This loss of extensive processivity is consistent with the observed motility properties of monomers (Ref. 7; but see also Refs. 32 and 33). Surprisingly, however, the kbi(ratio) of 4 indicates that even monomers of kinesin hydrolyze multiple ATP molecules during each productive encounter with a MT, unlike myosin S1 heads, for which the corresponding kbi(ratio) for interaction with actin is equal to 1 (34). This conclusion is supported by analysis of the transient kinetics of DKH357, which indicated that multiple hydrolytic cycles occur before diffusional separation of DKH357 from the MT (35).
The properties of DKH340 are unlike those of the other monomeric
constructs in a number of significant ways. The rate of release of ADP
from DKH340 in the absence of MTs (ko) is 3-5-fold more rapid than for the other constructs in both 25 mM KCl
(Table II) and 120 mM potassium acetate (Table III). In
contrast, the ko values for the other constructs,
both monomers and dimers, are very similar in each buffer. DKH340 also
has the highest kcat value for the maximum rate
at saturating levels of MTs (Table II) and the highest
kbi(ADP) value of the monomers (Table III). This
may represent an abnormally weak interaction with ADP that results in
both an elevated ko value in the absence of MTs and
an elevated kcat value at saturating MT levels.
A general facilitation of ADP release would also result in the
increased ability for low concentration of MTs to effect ADP release
and be responsible for the very low K0.5(MT)
value and high kbi(ADP) value of DKH340 compared
with the other monomers. Position 340 occurs at the junction of the
minimal motor unit and the predicted -helical neck domain. The
region on both sides of position 340 is highly conserved in all true
kinesins, and it may be that the C-terminal part of the motor domain
does not fold properly without part of the neck region. Energy-coupled
ATPases are unique in that their ATPase cycles have check points
through which they do not rapidly proceed unless some coupled event
such as interaction with a MT can occur. Incorrect folding of part of
the structure such as may occur in DKH340 could accelerate net ATPase
cycling if it selectively decreased the constraint that prevents
uncoupled ADP release. Another factor is that ADP release may be linked to conformational changes in the neck region that are energetically unfavorable. The absence of the neck domain in DKH340 would facilitate ADP release if this unfavorable conformational change could not occur
because there was no neck.
We thank J. Scholey for SUK4 cells; J. Nagey, S. Admiraal, B. Cobb, and T.-G. Huang for participation in preliminary phases of this work; and the Drug Synthesis and Chemistry Branch of the National Cancer Institute for Taxol.