 |
INTRODUCTION |
Ncd1 is a
microtubule-activated ATPase of the kinesin superfamily involved in
spindle assembly and stabilization during female meiosis and the early
divisions of the Drosophila embryo (1-4). Conventional
kinesin, the founding member of the kinesin superfamily, moves
membranous organelles from the neuron cell body to the synapse (reviewed in Refs. 5-7). The motor domains of Ncd and kinesin are
~40% identical in amino acid sequence, and their three-dimensional atomic structures are nearly superimposable (8-10). Despite this high
degree of similarity, there are striking differences in their motor
behavior that are important for biological function in vivo. First, Ncd translocates to the minus-end of microtubules, while kinesin
moves to the plus-end (2, 4, 11). Additionally, kinesin is a highly
processive motor, taking several steps per encounter with the
microtubule, while Ncd has been shown to be non-processive, both
mechanically and chemically (12-15). Ncd and kinesin also differ in
their motility and ATPase rates; Ncd is a much slower motor.
Comparison of the kinesin and Ncd crystal structures suggests that
despite the structural similarity of the individual catalytic motor
domains, there is a different overall orientation of the heads within
the Ncd dimer in comparison to those of the kinesin dimer, both in
solution and when bound to the microtubule (16-21). The neck linker, a
sequence of ~15 amino acids directly adjacent to the catalytic core,
has been implicated in specifying the direction of movement for kinesin
superfamily members and to be important for force generation (17,
22-24). For Ncd, the neck linker includes 13 amino acids N-terminal to
the catalytic core. There is high sequence homology among minus-end
directed kinesin motors, yet this neck linker sequence is different
from that of the plus-end directed kinesins. Furthermore, analysis of
kinesin-Ncd chimeric motors showed that the amino acid sequence of the
neck linker did indeed determine the polarity of microtubule movements
(22-24).
Recently published structural and spectroscopy studies by Rice et
al. (25) using monomeric kinesin K349 revealed a dramatic plus-end
directed conformational change in the neck linker upon ATP binding. The
neck linker became immobile and extended toward the microtubule
plus-end, yet the catalytic core did not shift its orientation on the
microtubule significantly. Furthermore, in the presence of ADP, the
neck linker returned to a state of high mobility comparable to solution
conditions in the absence of microtubules (K·ADP). These results with
monomeric kinesin K349 viewed within the context of the alternating
site mechanism of ATP hydrolysis (26-29) provide a plausible model for
kinesin plus-end directed motility and force generation.
Electron microscopy reconstructions have revealed that dimeric Ncd
binds the microtubule with its detached head pointed toward the
microtubule minus-end (17-21). This image is quite different from the
Mt·kinesin EM reconstructions and has been cited as a structural
intermediate important for Ncd minus-end directed movement. Our
experimental approach has been to explore the mechanistic features of
the Mt·Ncd ATPase that establish this intermediate and to account for
the structural transitions during the ATPase cycle to explain the
direction of Ncd motion and its non-processive motility.
Previous studies have defined a minimal kinetic mechanism for the
Mt·Ncd complex (14, 15, 30-34). It has been determined that ADP
release is the rate-limiting step in the mechanism. However, it is
still unclear how the mechanism of ATP hydrolysis for Ncd may relate to
its reversed directionality. Here we report that the two heads of
dimeric Ncd are not identical. Gel filtration and stopped-flow kinetics
reveal that the two heads within the dimer are different in solution in
the absence of microtubules, with one head binding ADP weakly and one
head binding ADP tightly. Stopped-flow experiments indicate that the
two heads of the dimer release ADP at different rates after interacting
with the microtubule. We propose that the head with the low affinity
for ADP binds the microtubule first to establish the intermediate
captured by cryo-EM with the detached head directed toward the
minus-end of the microtubule. The kinetics reveal cooperative
interactions within the dimer that account for the orientation of this
directionally biased Mt·Ncd intermediate. The kinetics also establish
a distinctive ATPase mechanism for Ncd and provide insight into the
mechanochemistry variability among kinesin superfamily members.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[
-32P]ATP (>3,000 Ci/mmol) was
purchased from PerkinElmer Life Sciences,
polyethyleneimine-cellulose F TLC plates (EM Science of Merck,
20 × 20 cm, plastic backed) from VWR Scientific (West Chester,
PA), and taxol (Taxus brevifolia) from Calbiochem. ATP, GTP,
DEAE-Sepharose FF, and SP-Sepharose were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). The
N-methylanthraniloyl derivatives of adenine nucleotides
(mant-ATP and mant-ADP) were synthesized and characterized as described
previously (15, 28, 35).
Buffer Conditions--
The following buffer was used for the
experiments described: ATPase buffer at 25 °C (20 mM
HEPES, pH 7.2, with KOH, 0.1 mM EDTA, 0.1 mM
EGTA, 5 mM Mg acetate, 50 mM K acetate, 1 mM dithiothreitol, 5% sucrose).
Protein Purification--
The dimeric Ncd construct (MC1) was
expressed in the Escherichia coli cell line BL21(DE3) from a
clone generously provided by Dr. Sharyn Endow, Duke University Medical
Center (36). This MC1 construct was expressed as a nonfusion protein
and contains amino acid residues Leu209-Lys700;
therefore, the N-terminal ATP-independent microtubule-binding site is
absent. MC1 was purified and characterized as described previously (15,
33, 36). Four different MC1 preparations were used for the
pre-steady-state experiments reported, and the steady-state parameters
were comparable to those reported previously: kcat = 2 s
1,
Km,ATP = 23 µM, and
K1/2,Mt = 20 µM. MC1 is dimeric
under the conditions of the experiments reported here based on the
Kd for dimerization for MC1 at <5 nM
(33). For the pre-steady-state experiments reported, we did not
pretreat the Mt·N complex with apyrase to remove bound ADP. This
approach was chosen due to concern that the Ncd protein behavior may be
altered by the treatment, leading to a significant fraction of inactive
protein associated with removal of the nucleotide (14, 32, 37). In
addition, one goal of the study was to understand cooperative
interactions between the two motor domains within the Ncd dimer.
Removal of nucleotide from the active sites of the dimer would render
the motor domains equivalent; therefore, the initial Ncd intermediate in the experiments may not be representative of the Ncd motor at the
same point in the ATPase cycle in vivo.
On the day of each experiment the microtubules were assembled from
soluble tubulin (cold depolymerized and clarified by centrifugation) and stabilized with 20 µM taxol as described previously
(38). This procedure yielded microtubules that were competent for
polymerization with essentially no soluble tubulin remaining.
All concentrations reported in the figure legends and manuscript
represent the final concentrations of the reactants after mixing.
Stopped-flow Kinetics--
The kinetics of mant-ATP binding and
mant-ADP release were measured using a KinTek Stopped-Flow Instrument
(Model SF-2001, KinTek Corp., Austin, TX) equipped with mercury arc
lamp at 25 °C in ATPase buffer. Each trace shown is an average of
4-8 traces. For experiments with the nucleotide analogs mant-ATP and
mant-ADP, the fluorescence emission at 450 nm was monitored using a
400-nm cut-off long-wave pass filter with excitation at 360 nm. The
ATP-promoted dissociation kinetics of the Mt·MC1 complex were
determined by turbidity measurements at 340 nm. The kinetic transients
were fit to the appropriate exponential functions (presented in figure legends) using KinTek software (version 6.00).
The stopped-flow transients in Fig. 3 were each fit to a single
exponential function plus a linear term to obtain the rate and the
amplitude of the observed process of mant-ADP release. The amplitudes
were plotted as a function of ATP concentration, and the data were fit
to a hyperbolic function (Fig. 3B). ATP
S (Fig.
3C), AMP-PNP (Fig. 3D), and AMP-PCP (data not
shown) were evaluated as competitive inhibitors of ATP. The fit of the
data to a hyperbola provided the K1/2,ATP in the
presence of either 50 µM ATP
S (Fig. 3C) or
1 mM AMP-PNP (Fig. 3D). The apparent Kd,ATP
S, and
Kd,AMP-PNP were obtained from Equation 1.
|
(Eq. 1)
|
Acid Quench Experiments--
The rate constant of ATP hydrolysis
was measured using a rapid chemical quench-flow instrument (KinTek
Corp.) at 25 °C in ATPase buffer. ATP hydrolysis was measured by
rapidly mixing the preformed Mt·N complex (1 µM MC1, 20 µM tubulin, 20 µM taxol, final after
mixing) with increasing concentrations of [
-32P]ATP.
The reaction was quenched with 2 N HCl and expelled from the instrument. Chloroform (100 µl) was immediately added and vortexed to denature the protein. The reaction was then neutralized with 2 M Tris, 3 N NaOH to pH 7.2-8.0.
An aliquot (1.5 µl) of each reaction mixture was spotted onto a
polyethyleneimine-cellulose TLC plate and subsequently developed with
0.6 M potassium phosphate buffer, pH 3.4, with phosphoric
acid. Radiolabeled nucleotide was quantified using a FUJI Bas-2000
PhosphorImager (Fuji Photo Film Co., Ltd). The acid quench released
bound nucleotide and products from the active site of MC1. Thus, the
product formed at each time point represents the sum of
N·ADP·Pi, N·ADP, and ADP released from the active
site of the enzyme and free in solution. In Scheme
1, we designate the radiolabeled
molecules by an asterisk (*). The data were analyzed by nonlinear
regression using KaleidaGraph software (Synergy Software, Reading,
PA).
Each time course of ATP hydrolysis was fit to the biphasic burst
equation,
|
(Eq. 2)
|
where A is the amplitude of the burst representing
the formation of [
-32P]ADP·Pi at the
active site; kb is the rate constant of the
pre-steady-state burst phase; kss is the rate constant of the linear phase and corresponds to steady-state turnover; and t is the time in seconds. The ATP concentration
dependence of the burst rate (Fig. 5B) and burst amplitude
(Fig. 5C) were fit to quadratic Equations 3 and 4,
respectively,
|
(Eq. 3)
|
|
(Eq. 4)
|
where kb is the rate of the exponential
burst; k4, the rate constant for ATP hydrolysis;
N0, the concentration of MC1 sites (1 µM); A, amplitude of the pre-steady-state
burst; Amax, maximum burst amplitude.
Gel Filtration to Determine Fraction ADP High Affinity
Sites--
MC1 is purified with ADP bound at the active site (33, 37);
therefore, the strategy of the assay is to use gel filtration to
quantify the concentration of [
32P]ADP that
partitions with MC1 protein. The centrifuge columns (Bio-Spin P-30
columns, 50-100-µl sample size, Bio-Rad Laboratories) were prepared
by washing the column initially with 500 µl of ATPase buffer
containing 0.25 mg/ml
-globulin (Bio-Rad protein standard), followed
by 3 washes (500 µl each) with ATPase buffer to equilibrate the
column and remove residual
-globulin that was added to decrease the
nonspecific binding of MC1 to the P-30 resin. A 300-µl reaction mixture containing 20 µM MC1 sites (estimated by
Bradford) was incubated at room temperature with 20 µM
[
32-P]ATP for 60-90 min to allow for ADP release,
followed by [
32-P]ATP binding and hydrolysis to label
the active motor sites. An 80-µl aliquot was applied to the
pre-washed centrifuge column (performed in duplicate) and centrifuged
at ~1000 × g for 5 min at 22 °C in a bench-top
swinging bucket centrifuge (2450 rpm, Sorvall RT 6000B Refrigerated
Tabletop Centrifuge). Approximately 80 µl (79-81.5 µl) was
recovered as the void volume for analysis by the Bradford Assay for
protein concentration and liquid scintillation counting for nucleotide
concentration determination. Aliquots of 5, 7, and 10 µl were used to
determine total counts for the calculation of nucleotide concentration,
and aliquots of 10 and 15 µl were used to determine protein
concentration. Parallel experiments were included as controls in which
either no protein was used in the reaction or dimeric kinesin K401 or
ovalbumin was used. These control experiments assessed the degree of
nonspecific binding of the MC1 to the gel filtration resin, whether all
free nucleotide partitioned within the bead pores, and whether there
were other inconsistencies in the assay procedure. Kinetic modeling and
simulations were performed using Scheme 1 with Scientist software
(MicroMath Scientific Software, Salt Lake City, UT).
 |
RESULTS |
ADP Release from Head 2--
The rate of mant-ADP release from the
Mt·MC1 complex was measured previously by rapidly mixing the
preformed MC1·mant-ADP complex with microtubules in the presence of
MgATP in the stopped-flow (15). A maximum observed rate of 3.7 s
1 was obtained, and the kinetics represent mant-ADP
dissociation from both motor domains. We then pursued experiments to
measure directly the kinetics of mant-ADP release from each motor
domain of the Ncd dimer. Cryo-EM studies have revealed a stable
Mt·Ncd intermediate in which one motor domain is bound to the
microtubule, yet the second motor domain is detached and directed
toward the minus-end of the microtubule (17-21). For our experiments,
we assumed that the detached motor domain would bind mant-ADP more
tightly than the motor domain bound to the microtubule (experimental
design shown in Fig. 1B)
because microtubules activate ADP release from 0.005 s
1
in the absence of microtubules to ~2 s
1 at high
microtubule concentrations (14, 15, 30-34, 37). To determine the rate
of mant-ADP release from the detached motor domain (head 2),
microtubules, MC1, and mant-ADP were preincubated to form the
Mt·MC1·mant-ADP complex (1 mant-ADP per MC1 dimer). This complex
was rapidly mixed with MgATP to initiate mant-ADP release from head
2.

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 1.
Mant-ADP release from head 2 initiated by
MgATP. The Mt·N·mant-ADP (1 µM MC1, 0.5 µM mant-ADP, 20 µM tubulin) was preformed
with limiting mant-ADP with the assumption that mant-ADP would
partition to the ADP high affinity site of MC1 and the low affinity
site (head bound to the microtubule) would be unoccupied. The
experimental design is shown in the inset of Panel
B. The Mt·N·mant-ADP complex was rapidly mixed in the
stopped-flow instrument with varying concentrations of MgATP (1-100
µM). MgATP binding at the empty microtubule-bound site
stimulates release of mant-ADP from the high affinity site (head 2).
A, transients are shown for various ATP concentrations: 1, 2, 3, 5, 10, 20, 30, 50, and 100 µM ATP (from top to
bottom transient). Each transient was fit to a single exponential
function plus a linear term. B, the rate constant obtained
from the exponential phase plotted as a function of microtubule
concentration. Fit of the data to a hyperbola yields
kobs = 1.4 ± 0.02 s 1 with
half-maximal stimulation occurring at 0.64 ± 0.06 µM ATP.
|
|
Fig. 1 shows the time dependence of the fluorescence change as mant-ADP
is released from the more hydrophobic active site of the motor domain
to the solution where the fluorescence is quenched. Both the rate and
the amplitude associated with the fluorescence exponential phase
increases as a function of ATP concentration. The rate of mant-ADP
release from head 2 is ATP concentration dependent with the maximum
rate constant at 1.4 s
1 (K1/2 = 0.6 µM ATP). These results suggest that ADP release from head 2 is rate-limiting for steady-state turnover because all other
steps in the pathway have been determined to be significantly faster
than the kcat at 2 s
1 (14, 15).
This experiment also implies that ATP binding at the first head is
necessary for the second head to bind the microtubule and release its
mant-ADP.
Fig. 2 presents the same experiment but
with mant-ADP release from head 2 initiated by MgADP. The maximum rate
constant observed was 1.3 s
1 (K1/2 = 0.5 µM ADP). Note that the ADP-promoted kinetics of
mant-ADP release were comparable to those observed for ATP (Fig. 1),
both for the observed rate of mant-ADP dissociation as well as the K1/2. These data suggest that the conformation
required for mant-ADP release from head 2 can be achieved either by ATP
binding and hydrolysis at head 1 or by ADP binding directly to head 1 to induce the structural transition.

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 2.
ADP promoted mant-ADP release from head
2. The Mt·N·mant-ADP complex (1 µM MC1, 0.5 µM mant-ADP, 20 µM tubulin) was preformed
as described in the legend to Fig. 1 such that one mant-ADP was bound
per MC1 dimer. The Mt·N·mant-ADP complex was rapidly mixed in the
stopped-flow instrument with varying concentrations of MgADP (1-1000
µM). A, transients are shown for various ADP
concentrations: 1, 1.5, 3, 5, 10, 25, and 50 µM (from top
to bottom transient). Each transient was fit to a single exponential
function plus a linear term. B, the rate constant obtained
from the exponential phase plotted as a function of microtubule
concentration. Fit of the data to a hyperbola yields
kobs = 1.3 ± 0.02 s 1 with
half-maximal stimulation occurring at 0.51 ± 0.06 µM ADP.
|
|
Is ATP Hydrolysis Required for Head 2 Mant-ADP Release?--
This
experiment was performed using several nucleotides and nucleotide
analogs to determine whether the post-ATP hydrolysis state at head 1 is
required for the second head to bind the microtubule and release its
mant-ADP (Fig. 3). The Mt·N·mant-ADP
complex (1 mant-ADP per MC1 dimer) was rapidly mixed with buffer, ATP,
ADP, the non-hydrolyzable ATP analogs, AMP-PNP and AMP-PCP, or the slowly hydrolyzable ATP analog, ATP
S. In the presence of 1 mM ATP and ADP, mant-ADP is released and the amplitude
associated with the kinetics is significantly larger than the amplitude
associated with the kinetics at the other experimental conditions. In
the buffer control and thus in the absence of added nucleotide, the amplitude of mant-ADP release is <5% of the amplitude change
associated with ATP or ADP. Although the amplitude of the AMP-PCP
kinetics is also quite low, subsequent experiments revealed that
AMP-PCP does not compete with ATP for binding MC1 active sites. In the presence of 1 mM AMP-PNP and ATP
S, the amplitude of the
fluorescence change is less than 24% of the amplitude change observed
in the presence of 1 mM ATP or ADP. Yet, both AMP-PNP and
ATP
S bind to the active site as effectively as either ADP or ATP at
1 mM analog concentration (apparent
Kd,AMP-PNP = 199 µM and
Kd,ATP
S = 1.1 µM). These data indicate that ATP binding at head 1 is
not sufficient for dissociation of the second high affinity mant-ADP,
and either the ADP·Pi or ADP state is required for the
second motor domain to bind the microtubule and release its mant-ADP.
We attribute the fluorescence change in the presence of AMP-PNP and
ATP
S to the fact that the analogs cannot exactly form the
Mt·N·ATP intermediate conformation, and AMP-PNP and ATP
S may in
fact resemble an ADP·Pi intermediate to a limited extent.
The small amplitude associated with the AMP-PNP and ATP
S promoted
kinetics of mant-ADP dissociation is consistent with the interpretation
that a post-ATP hydrolysis intermediate (either the
ADP·Pi or ADP state) at head 1 is necessary for the
second head to bind the microtubule and release its tightly bound
mant-ADP.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 3.
Mant-ADP release from head 2 in the presence
of non-hydrolyzable or slowly hydrolyzable ATP analogs. The
preformed Mt·N·mant-ADP complex (1 µM MC1, 0.5 µM mant-ADP, 20 µM microtubules) was
rapidly mixed in the stopped-flow with buffer, Mg·AMP-PCP,
Mg·AMP-PNP, Mg·ATP S, MgATP, or MgADP. Experimental design as
depicted in Figs. 1-3B. A, the observed decrease
in mant-ADP fluorescence as a function of 1 mM nucleotide
concentration. The relative amplitude associated with the ATP S and
AMP-PNP-promoted transients was 24% of that associated with ATP and
ADP experiments. The AMP-PCP and buffer transients showed no
significant change in the fluorescence upon mixing. B, the
change in relative fluorescence amplitude plotted as a function of ATP
concentration. The transients and exponential rates for this experiment
were presented in Fig. 1. The fit of the data to a hyperbola provided
the maximum ATP-dependent amplitude of 0.88 ± 0.031 with an apparent Kd,ATP = 6.98 ± 0.90 µM. The inset is the schematic of the
experimental design. C, the change in relative fluorescence
amplitude as a function of ATP concentration in the presence of 50 µM Mg·ATP S. The fit of the data to a hyperbola
yielded a maximum amplitude of 0.85 ± 0.06 with an observed
K1/2,ATP = 319 ± 78 µM. The
apparent binding constant for ATP S was determined using Equation 1
which yielded Kd,ATP S = 1.1 µM. The inset presents the stopped-flow
transients where the top transient is the reaction with 50 µM ATP S in the absence of ATP, and the bottom
trace the reaction with 100 µM ATP in the absence of
ATP S. The intermediate transients (from top to bottom) all contain
ATP S at 50 µM with varying ATP concentration (25, 100, 1000, and 5000 µM ATP). D, the change in
relative fluorescence amplitude as a function of ATP concentration in
the presence of AMP-PNP. This experiment, complementary to
C, shows the kinetics of ATP-promoted mant-ADP release in
the presence of 1 mM AMP-PNP. The amplitude of the
exponential phase of each transient was plotted as a function of ATP
concentration. The fit of the data to a hyperbola yielded a maximum
amplitude of 1.20 ± 0.04 with an observed
K1/2,ATP = 42.0 ± 4.8 µM.
Equation 1 provided the apparent
Kd,AMP-PNP = 199 µM. The
inset presents the individual stopped-flow transients where
the top transient shows the reaction with 1 mM AMP-PNP in
the absence of MgATP and the bottom transient, the reaction with 1 mM MgATP in the absence of AMP-PNP. The intermediate
transients (from top to bottom) all contain AMP-PNP at 1 mM
with varying MgATP concentrations (5, 20, 50, and 1000 µM
ATP).
|
|
ADP Release from Head 1--
In order to determine the kinetics of
mant-ADP dissociation from the first motor domain, MC1 was incubated
with mant-ADP to replace ADP bound at the active sites of the dimer (2 mant-ADP per MC1 site, 4 mant-ADP per dimer). The MC1·mant-ADP
complex was then rapidly mixed with microtubules in the absence of
added nucleotide in the stopped-flow. In the absence of added ATP or ADP, microtubule-activated mant-ADP release from head 1 only is observed (Fig. 3). Fig. 4 shows the time
dependence of the fluorescence change at eight different microtubule
concentrations. The rate of the initial exponential phase increased as
a function of microtubule concentration, and the fit of the data to a
hyperbola yielded the maximum rate of mant-ADP release from the first
head at 18 s
1. This rate is significantly faster than the
rate of mant-ADP release observed for the second motor domain at 1.4 s
1 (Fig. 1). These data are consistent with the model
shown in Scheme 1 in which rapid ADP release from head 1 is followed by
ATP binding and hydrolysis at this site, causing the second motor
domain to bind the microtubule and release its mant-ADP in the
rate-limiting step of the cycle.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 4.
Mant-ADP release from head 1. The
N·mant-ADP complex (6 µM MC1, 12 µM
mant-ADP) was preformed at a 1:2 ratio of MC1·mant-ADP to exchange
the ADP for mant-ADP at both sites of the MC1 dimer (inset panel
B). This complex was rapidly mixed in the stopped-flow instrument
with varying concentrations of microtubules (1-20 µM
tubulin). The experimental design assumes that in the absence of ATP or
ADP (Fig. 3), mant-ADP will be released from head 1 only. A,
transients shown are for the following microtubule concentrations: 0, 1, 3, 6, 8, 10, 16, and 20 µM tubulin (from top to bottom
transient). Each transient was fit to a single exponential function
plus a linear term. Mant-ADP release in the absence of microtubules is
0.005 s 1. B, the rate constants obtained from
the exponential phase plotted as a function of microtubule
concentration. Fit of the data to a hyperbola yields
kobs = 17.8 ± 1.6 s 1 with
half-maximal stimulation occurring at 2.2 ± 2.0 µM
tubulin.
|
|
Tight Binding of ATP by One Head--
The kinetics of mant-ADP
release activated by either ATP or ADP revealed a
K1/2 of 0.5-0.6 µM (Figs. 1 and
2). These results showed that mant-ADP release from head 2 was
activated at very low ATP concentrations which appeared surprising
based on the Km,ATP at 23 µM determined by steady-state kinetics and the
Kd,ATP at 16 µM determined
by the rapid quench experiments (15, 33). However, the head 2 mant-ADP
release kinetics reflect ATP binding and hydrolysis to head 1 only
while the steady-state and acid quench kinetics evaluate ATP turnover
at both motor domains. Furthermore, because the sensitivity of the
fluorescence signal is so high, very low concentrations of ATP could be
used to evaluate mant-ADP release (Fig. 1). The mant-ADP release
kinetics revealed a tight site for ATP binding that was not evident in
our earlier experiments because their experimental design evaluated the
composite behavior of both ATP-binding sites of the Ncd dimer, a tight
site and a weak site.
We pursued acid quench experiments with MC1 to examine ATP binding and
hydrolysis by head 1 at very low ATP concentrations, and these results
are presented in Fig. 5. The time course
for ATP hydrolysis was measured at 1 µM MC1 to examine
the kinetics at significantly lower ATP concentrations than performed
previously (15). Fig. 5A shows transients at four different
ATP concentrations (2, 5, 10, and 100 µM ATP). There was
an initial exponential burst of product formation corresponding to the
formation of the N·ADP·Pi intermediate during the first
turnover, followed by a slower linear phase which
represents subsequent ATP turnovers. The rate constants determined for
the linear phase of each transient were consistent with those
determined by steady-state kinetics at 20 µM microtubules (kcat = 1.1 ± 0.03 s
1;
Km,ATP = 16.9 ± 2.3 µM).

View larger version (73K):
[in this window]
[in a new window]
|
Fig. 5.
Pre-steady-state ATP hydrolysis by MC1.
The preformed Mt·N complex (1 µM MC1, 20 µM tubulin) was rapidly mixed in the chemical quench flow
instrument with varying concentrations of [ 32P]MgATP
and allowed to react for 0.01-2 s as described under "Experimental
Procedures." All concentrations reported are final after mixing.
A, the transients for ATP hydrolysis in the presence of 2 µM ( ), 5 µM ( ), 10 µM
( ), and 100 µM ( ) [ 32P]MgATP.
The data were fit to the burst equation (Equation 2). B, the
pre-steady-state kinetic burst rate constant calculated for each
transient and plotted as a function of [ 32P]MgATP
concentration. The data were fit to Equation 3 which yields
k+4 = 35.4 ± 5.0 s 1 and
Kd,ATP = 3.4 ± 1.5 µM. The [ 32P]MgATP concentrations used
were sufficiently high such that ATP binding
(k+3) no longer limits the rate of the first
turnover. Therefore, the maximum rate constant for the burst predicts
the maximum rate constant for ATP hydrolysis
(k+4). C, the amplitude of the
pre-steady-state burst plotted as a function of
[ 32P]MgATP concentration. The data were fit to
Equation 4: maximum burst amplitude = 0.81 ± 0.06 µM with the Kd,ATP = 17.8 ± 3.5 µM. Panels B and C
contain data from experiments not shown in A.
|
|
The rate of the initial exponential phase increased as a function of
ATP concentration (Fig. 5B), and the data were fit to Equation 3. The maximum rate of ATP hydrolysis was 35 s
1
and the Kd,ATP was 3.4 µM.
The burst amplitude at 40 µM ATP (Fig. 5C) was
0.5 µM (~50% of the enzyme site concentration), indicating that the data obtained at these lower ATP concentrations represents only one head. The maximum burst amplitude obtained from the
fit of the data was 0.8 µM and thus representing ATP hydrolysis at both motor domains of the dimer. The
Kd,ATP at 17.8 µM
determined from the burst amplitude data (Fig. 5C) is
similar to the Kd,ATP reported
previously at 15 µM (15). These results indicate that the
rate constants for ATP hydrolysis at head 1 (Scheme 1,
k4) and head 2 (k8) are
similar, yet the ATP binding affinities at each site differ. The
Kd,ATP obtained from panel B
represents the Kd,ATP for head 1 and
suggests this site binds ATP tightly. The
Kd,ATP determined from the burst
amplitude data (Fig. 5C) represents both sites of the dimer,
implying that the second ATP molecule binds more weakly than the first.
The burst amplitude at 0.8 µM rather than 1 µM is attributed to the loss of signal at very high ATP
concentrations in rapid quench-flow experiments. We reported previously
that the maximum burst amplitude was approximately equal to the site
concentration used in the experiment (15).
Asymmetry within the Ncd Dimer--
Early experiments suggested
that the two sites of MC1 free in solution bound ADP with different
affinities. We tested this hypothesis directly by rapidly mixing
mant-ATP with dimeric MC1 in the stopped-flow in the absence of
microtubules (Fig. 6). The kinetics of
mant-ATP binding revealed a rapid exponential burst of fluorescence
enhancement associated with mant-ATP binding to the active site,
followed by a significantly slower linear phase at 0.005 s
1. The rate of the exponential phase increased as a
function of mant-ATP concentration, and the fit of the data to a
hyperbola provided the maximum rate at 7 s
1. The
observation of the exponential burst in this experiment is indicative
that there were MC1 sites unoccupied and available to bind mant-ATP
immediately. If ADP were tightly bound at all MC1 active sites, the
kinetics of mant-ATP binding would appear linear and reflect the slow
release of ADP at <0.01 s
1 as observed in the linear
phase of the transient in Fig. 6 and reported previously for MC1
(15).

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 6.
MantATP binding to MC1 in the absence of
microtubules. A, MC1·ADP as purified (10 µM) was rapidly mixed in the stopped-flow instrument with
20 µM mant-ATP. The jagged line represents the
fluorescence enhancement, and the smooth line is the fit of
the data to the burst equation (Equation 2). The rate of the
exponential phase was 3.6 ± 0.4 s 1, and the rate of
the linear phase was 0.005 ± 0.00006 s 1.
B, the observed exponential rate constant was plotted as a
function of mant-ATP concentration. The data were fit to a hyperbola
with a maximum rate constant of the first-order observed process at
7.0 ± 0.6 s 1. Experiments at lower mant-ATP
concentrations were performed with 5 µM MC1 (5-10
µM mant-ATP) and 1 µM MC1 (1-5
µM mant-ATP).
|
|
Although this stopped-flow experiment revealed unoccupied active sites,
the amplitude of the fluorescence signal is relative and cannot be
directly correlated with the concentration of MC1 active sites used in
the experiment. Gel filtration experiments were performed to quantify
the concentration of [
32P]ADP that partitions with
MC1 protein. In this experiment, MC1 was incubated with
[
32P]ATP for sufficient time to allow ADP to be
released from the active site and radiolabeled ATP to bind and be
hydrolyzed. The samples were then applied to a gel filtration column
and centrifuged. The concentrations of MC1 and
[
32P]ADP were determined for the excluded volume.
The results in Table I show that the
concentration of radiolabeled ADP that partitioned with MC1 was
approximately half the concentration of MC1 protein (0.6:1). In
contrast, the concentration of radiolabeled ADP that partitioned with
dimeric kinesin K401 was 0.9:1 and for ovalbumin, 0.035:1. These
results suggest that within the Ncd dimer, the active sites bind ADP
with different affinities with one site binding ADP tightly and the
other site binding ADP more weakly. The alternative interpretation that
50% of the MC1 protein is inactive appears unlikely. The protein
concentration determined by the Bio-Rad protein assay was comparable to
the concentration of active sites determined by acid-quench burst
experiments and the creatine kinase active site titration (15, 34).
These results indicate that one motor domain binds ADP tightly while the other head binds ADP more weakly, resulting in a rapid equilibrium at this weak site. These data suggest that upon dimerization, an
asymmetry is established between the motor domains, and this asymmetry
is intrinsic to the dimer before it interacts with the microtubule. Note that this asymmetry was not observed in conventional kinesin K401.
ATP-promoted Dissociation Kinetics--
As we began to evaluate
different models for the Mt·Ncd ATPase mechanism, the need to
understand the point in the cycle in which the Ncd dimer completely
detaches from the microtubule became critical. Previously, the rate
constant for dissociation was reported at 13 s
1 (15), yet
it was unclear whether detachment of head 1 occurred at 13 s
1 or whether the 13 s
1 represented the
dissociation of the dimer from the microtubule. As the dissociation
kinetics were re-evaluated, it was apparent that the kinetics were
biphasic with a rapid exponential phase, followed by a second,
significantly slower exponential phase (Fig. 7). We tested the hypothesis that the
initial fast exponential phase represented detachment of head 1, and
the second, slow exponential phase of the turbidity kinetics
represented dissociation of head 2. The experiments were repeated to
analyze both exponential phases as a function of ATP (Fig. 7). As
observed previously, the rate of the initial exponential phase
increased as a function of ATP and was fast with the maximum observed
rate constant at 12 s
1. The rate of the second
exponential phase also increased as a function of ATP concentration
with the maximum observed rate of dissociation at 1.4 s
1.
These results are consistent with sequential detachment of the motor
domains (Scheme 1). Although the dissociation kinetics in the second
phase were observed at 1.4 s
1, this rate constant does
not necessarily represent the intrinsic rate constant. The mant-ADP
release kinetics in Fig. 1 indicated that ADP release occurred at 1.4 s
1 and was rate-limiting for the pathway; therefore, any
step that occurs after this slow step (k6) will
be limited by the 1.4 s
1 event and observed
experimentally at no faster than 1.4 s
1.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 7.
ATP-promoted dissociation kinetics of the
Mt·MC1 complex. Panel A shows a representative
stopped-flow transient in which the Mt·MC1 complex (5 µM MC1 and 4.9 µM tubulin, 20 µM taxol) was mixed with 700 µM MgATP
(final concentrations after mixing). The smooth line shows
the fit of the data to a double exponential function with the initial
fast phase at 12.2 ± 0.3 s 1 and the second slower
phase at 1.2 ± 0.05 s 1. The inset shows
the ATP concentration dependence of the initial fast exponential phase.
The fit of the data to a hyperbola provides the maximum rate constant
at 12.2 ± 0.4 s 1 which is comparable to our
previously reported dissociation rate constant (15). Panel B
presents the ATP concentration dependence of the second, slower
exponential phase with the maximum rate constant at 1.4 ± 0.07 s 1. Included in Panel B is a schematic of the
experimental design. Experiments were performed at three different
concentrations of Mt·MC1 complex to optimize the turbidity signal: 5 µM MC1 + 4.9 µM tubulin for 5-500
µM MgATP, 2.5 µM MC1 + 2.3 µM
tubulin for 2.5-50 µM MgATP, and 1 µM MC1 + 1 µM tubulin for 0.5-5 µM MgATP.
|
|
 |
DISCUSSION |
Asymmetry within the Ncd Dimer--
The mant-ATP binding kinetics
and gel filtration experiments (Fig. 6, Table I) clearly indicate two
sites exhibiting different affinities for ADP, one binding ADP weakly
while the other binds ADP tightly. These results were really surprising
because the two polypeptides are equivalent in amino acid sequence,
length, and presumably state of post-translational modification because the protein is expressed in an E. coli expression system
from a single gene. Furthermore, this behavior was never seen with the
kinesin dimer, K401 either in the control experiments presented here or
in the nitrocellulose binding assays published previously (38). These
data for MC1 suggest that upon dimerization, an asymmetry is
established between the motor domains, and this asymmetry is intrinsic
to the dimer before it interacts with the microtubule.
The kinetic and gel filtration data presented here would appear to be
in direct conflict with the Ncd dimeric crystal structure showing both
active sites occupied by ADP (17). However, the crystallization
conditions included 2 mM MgADP to stabilize the protein.
Our results are completely consistent with the structural studies
because the addition of 2 mM MgADP is expected to drive the
equilibrium toward dimeric Ncd with both the weak and tight sites
occupied by ADP. The mechanistic experiments presented here (Fig. 6,
Table I) have revealed a structural intermediate that has not been
detected previously and may not be detectable by conventional imaging
and crystallography approaches because Ncd is labile and degrades in
the absence of ADP.
The ATPase Pathway--
Scheme 1 shows our model for the Mt·Ncd
ATPase based on the equilibrium binding studies (33), the
pre-steady-state kinetics (14, 15, 34), the motility (13, 17, 24), and
structural results for Ncd (16-21, 39, 40). The cycle begins at the
star (
) intermediate, and the experimentally determined rate
constants are designated. We propose that the motor domain that holds
ADP more weakly (designated head 1) binds the microtubule first and stimulates fast release of ADP. The asymmetry in the Ncd dimer establishes the Mt·Ncd intermediate observed in the cryo-electron micrographs in which one motor domain is bound to the microtubule with
the second motor domain detached and pointed toward the minus-end of
the microtubule (upper right, intermediate 3). ATP then binds to the
empty site (head 1), followed by rapid ATP hydrolysis which is required
for head 2 to bind to the microtubule. (Rapid quench radiolabeled
species are indicated by the asterisk.) Head 1 detaches from the
microtubule as the N·ADP·Pi intermediate with the
second head poised for rate-limiting ADP product release at 1.4 s
1. A second round of ATP binding and hydrolysis is
required to release head 2 and therefore release the dimer from the
microtubule. This model is consistent for a non-processive dimeric
motor and provides a mechanism for directional bias intrinsic to the
Ncd dimer. Furthermore, this model predicts that dimeric Ncd takes a
single step to the next microtubule binding site, yet 2 ATP molecules
are required for this step and the force-generating structural
transitions for minus-end directed movement.
There were several key experiments that excluded other potential
models. The experiment presented in Fig. 1 was designed to begin as
intermediate 3 with mant-ADP bound at the high affinity site. The slow
rate of mant-ADP release at 1.4 s
1
(k6) is the slowest step measured in the pathway
and is therefore rate-limiting for steady-state turnover. Furthermore,
the results presented in Fig. 3 established that ATP hydrolysis at head
1 (k4) to reach the ADP·Pi or ADP
state was required for mant-ADP release from head 2 (k6). Thus, the results in Figs. 1-3 revealed the intermolecular cooperativity that was required for rate-limiting mant-ADP release.
The equilibrium binding experiments published previously (33) indicated
that the only conditions that led to Ncd partitioning off the
microtubule were ADP + Pi, indicating that the
N·ADP·Pi intermediate was the nucleotide intermediate
that detached from the microtubule. Furthermore, these data were
sigmoidal, and the fit to the Hill equation indicated that two sites
were cooperative. These results as well as the rapid quench burst
amplitude data presented here (Fig. 5) and in Ref. 15 are indicative
that both sites must hydrolyze ATP before the Ncd dimer is released
from the microtubule (k9). Therefore, these
experiments suggest an ATPase cycle in which both motor domains of the
dimer must participate directly.
Our interpretation of the biphasic dissociation kinetics presented in
Fig. 7 requires the assumption that the turbidity signal associated
with intermediate 3 is greater than intermediate 6, and both are
greater than the turbidity signal of the microtubule with Ncd detached
and free in solution. Although at first glance this assumption may seem
naive, there are several lines of evidence that support the
interpretation that the second phase of the turbidity kinetics
represents a true step on the pathway. Experimentally, we cannot
determine a rate constant for head 2 detachment any faster than 1.4 s
1 because this step is limited by ADP release at
k6 = 1.4 s
1. The fact that the
second phase of the dissociation kinetics is ATP-dependent
is indicative that this exponential phase represents a true step on the
pathway rather than a nonspecific, slow linear phase seen at the end of
stopped-flow transients. These are typically very slow and not ATP
dependent. Furthermore, the rapid quench burst experiments show that
both motor domains hydrolyze ATP during the exponential burst phase and
prior to steady-state (Fig. 5). Last, the ATP-promoted dissociation
kinetics for monomeric Ncd, MC6, reveal that the monomer does not
detach from the microtubule. ATP-promoted dissociation requires a
dimeric Ncd motor (34).
A very careful mechanistic study on dimeric Ncd was published by
Pechatnikova and Taylor (14), and our kinetics are very similar to
theirs. However, we have excluded their model based on our previously
published dissociation kinetics (33). In the Pechatnikova and Taylor
model, ATP binding at the vacant site of intermediate 3 (our Scheme 1)
leads to detachment of Ncd as the ATP·N·ADP intermediate. This
intermediate subsequently rebinds to the microtubule by the
ADP-containing head, followed by ATP hydrolysis, and ADP release as the
rate-limiting step. We propose that Ncd cannot detach as the
ATP·N·ADP intermediate because ATP hydrolysis is required for
dissociation (33). We performed a dissociation experiment as shown in
Fig. 7, but dissociation was initiated by either ATP or the
nonhydrolyzable ATP analog, AMP-PNP. In the presence of AMP-PNP, there
was no change in the turbidity signal indicating that ATP binding is
not sufficient to stimulate dissociation. These kinetics show that ATP
binding and ATP hydrolysis must both occur for Ncd to detach from the
microtubule (33).
The model presented in Scheme 1 is consistent with the mechanistic,
motility, and structural results for dimeric Ncd. This model accounts
for the minus-end direction of motion and reveals cooperative
interactions that are important for force generation for a
non-processive dimeric motor. This model is attractive because it
provides a mechanism to account for the directional bias of intermediate 3 and minus-end directed Ncd motility. The determinants for minus-end directionality have been localized to the neck linker sequence, and these results evaluated in the context of the kinetics presented here lead to the testable hypothesis that the neck linker sequence establishes the asymmetry within the Ncd dimer. The sequential ATPase mechanism for Ncd is quite different from conventional kinesin's ATPase mechanism. This study with dimeric Ncd illustrates the mechanistic diversity for energy transduction that is utilized by
two kinesin superfamily members.