From the Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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Full-length Drosophila kinesin heavy
chain from position 1 to 975 was expressed in Escherichia
coil (DKH975) and is a dimer. The sedimentation coefficient of
DKH975 shifts from 5.4 S at 1 M NaCl to ~6.9 S at <0.2
M NaCl. This transition of DKH975 between extended and
compact conformations is essentially identical to that for the heavy
chain dimer of bovine kinesin (Hackney, D. D., Levitt, J. D.,
and Suhan, J. (1992) J. Biol. Chem. 267, 8696-8701). Thus the capacity for undergoing the 7 S/5 S transition is an intrinsic
property of the heavy chains and requires neither light chains nor
eukaryotic post-translational modification. DKH960 undergoes a similar
transition, indicating that the extreme COOH-terminal region is not
required. More extensive deletions from the COOH-terminal (DKH945 and
DKH937) result in a shift in the midpoint for the transition to lower
salt concentrations. DKH927 and shorter constructs remaining extended
even in the absence of added salt. Thus the COOH-terminal ~50 amino
acids are required for the formation of the compact conformation.
Separately expressed COOH-terminal tail segments and
NH2-terminal head/neck segments interact in a
salt-dependent manner that is consistent with the compact
conformer being produced by the interaction of domains from these
regions of the heavy chain dimer. The microtubule-stimulated ATPase
rate of DKH975 in the compact conformer is strongly inhibited compared
with the rate of extended DKH894 (4 s Kinesin is an ATP-dependent motor protein that is
involved in movement of membranous vesicles along
MTs1 (see Refs. 1 and 2). The
NH2-terminal ~340 amino acids of the heavy chain forms a
globular motor domain (head) that has MT-stimulated ATPase activity
(see Fig. 2B). The motor domain is followed by a long
central coiled-coil stalk region and a small nonhelical domain at the
COOH-terminal. The central region contains several positions at which
the coiled-coil propensity is low and these likely represent hinges in
the stalk. The first coiled-coil region that extends from the motor
domain is designated the neck region. Constructs that contain the head
and the COOH-terminal part of the coiled-coil neck form dimers (3).
There is a likely hinge at position ~400 that marks the boundary
between the neck and the stalk. Peptides from the neck (4, 5) and stalk
(6) have been demonstrated to interact in a coiled-coil manner by several criteria. The crystal structure of a dimeric head plus neck
construct has been recently determined (7) and it directly demonstrates
the coiled-coil interactions in the neck region.
Native kinesin is a heterotetramer composed of a dimeric heavy chain
core with two light chains attached in the COOH-terminal region (8, 9).
At high salt kinesin exist in an extended conformation with an
s20,w value of ~6 S, but adopts a more
compact conformation at low salt concentration with an s20,w value of ~9 S. This global
conformational transition is readily reversible and can be observed by
electron microscopy (10, 11). A species lacking detectable light chains
is also obtained from bovine brain and undergoes a corresponding
salt-dependent transition between extended (~5 S) and
compact (~7 S) conformations (11). We report here that
Drosophila heavy chain dimers also undergo a 7 S/5 S
transition. Furthermore, specific regions of the tail and head/neck
interact in a salt-dependent manner that is consistent with
this interaction being responsible for producing the compact conformation.
Isolated head domains have a high MT-activated ATPase activity when
prepared by limited proteolysis (12) or as fusion proteins (13). The
rate of ~40 s It has not, however, been possible to directly test the inhibition of
ATPase of kinesin in the compact versus the extended conformers because the transition could only be produced at high ionic
strength. MT-stimulated ATPase and MT-stimulated ADP release are
strongly inhibited by even moderate salt concentrations (18, 19) and it
would be difficult to separate the direct inhibitory effect of salts
from their possible secondary activation linked to the 7 S/5 S
transition. We report here that removal of a small COOH-terminal region
blocks formation of the compact conformation. This allows comparison of
the MT-stimulated ATPase rate of full-length kinesin in the compact
conformation to that of this slightly shorter extended construct at low
salt where both are potentially active. Under these conditions, the
extended construct has the same high ATPase activity as short dimers of
heads, whereas the compact construct is strongly inhibited.
All reactions were performed at 25 °C in A25 buffer as
described previously (3). Centrifugation on sucrose density gradients and gel filtration on Bio-Gel A-5m were performed using standard proteins as described previously (11). SDS-PAGE was performed by the
method of Laemmli (20) using a 4% stacking gel and a 11% separating
gel, except for the separation of the long constructs (Fig.
1B) which used a 6% separating gel.
Construction of Expression Plasmids--
All kinesin constructs
were derived from the original Drosophila cDNA clone of
Yang et al. (21). pGST864-975 encodes the tail domain
region between positions 864 and 975 as a fusion protein with GSTase.
It was obtained by ligation of the PvuII fragment of kinesin
that contains the COOH-terminal into the SmaI site of
pGEX-3X (Pharmacia). pGST864-893 was obtained by cleavage of pGST864-975 with NcoI and EcoRI, treatment with
DNA polymerase (Klenow), and religation. This procedure removes the
coding region between amino acids 894 and the COOH terminus and results
in a fusion protein with an extension of EFIVTD beyond position 893. pGST893-975 was obtained by insertion of the
NcoI/EcoRI fragment of kinesin that contains the
COOH-terminal into a modified pGEX-2T (Pharmacia) that contains an
NcoI site following the BamHI site of the
multiple cloning site. The resulting construct contains pGEX-2T
sequence through the thrombin cleavage site terminating in PRGS,
followed by PA and then kinesin sequence from Met893 to the
COOH-terminal. This was confirmed by DNA sequencing. pGST613-975 was
similarly obtained by insertion of the NcoI/EcoRI
fragment of kinesin that was not cut at the internal NcoI
site at 893. Constructs terminating at 910, 937, and 960 were obtained
by polymerase chain reaction using pDK613-975 (see below) as the
target and with T7 promoter primer in the forward direction and reverse
primers that introduced a stop codon at the appropriate position
followed by an EcoRI site. These polymerase chain reaction
products were cleaved with XhoI and EcoRI and
inserted into a backbone derived from pGST613-975 by cleavage with
XhoI and EcoRI. This produced a set of constructs
in pGEX with kinesin sequence from 613 to the new stop positions.
pGST893-910, pGST893-937 and pGST893-960 were produced from these
constructs by cleavage with NcoI and religation of the
backbone to remove the 613-892 region.
pDKH975 contains the entire coding region of Drosophila
kinesin heavy chain from position 1 to 975 as a non-fusion protein. It
was derived from pDKH392 (22) by cleavage with NsiI and
EcoRI and insertion of the NsiI to
EcoRI fragment of kinesin. pDKH894, pDKH910, pDKH927,
pDKH937, pDKH945, and pDKH960 code for amino acids from position 1 to
the indicated stop position. They were produced by insertion into
pDKH975 of either the NcoI/EcoRI fragment from
pGST893-910, pGST893-937, and pGST893-960 or of new polymerase chain
reaction fragments. In the final constructs, the only coding region
that is polymerase chain reaction-derived is between the NcoI site at Met893 and the stop codon and this
was confirmed by DNA sequencing. pDKH614 was derived from pDKH894 by
cleavage with NcoI and religation of the backbone. In
pDKH614, Ala614 is converted to Glu. pDKH560 was derived
from pDKH614 by cleavage with NsiI and NcoI and
insertion of the NsiI/AflIII fragment of kinesin.
In pDKH560, Ser560 is converted to Glu. pDK613-975 and
pDK893-975 contain the indicated kinesin regions as non-fusion
proteins in pET-21d (Novagen).
Protein Expression and Purification--
Kinesin head domains
were prepared as described previously (3, 14). The long heavy chain
constructs, DKH894-DKH975, were expressed and processed through the
P-11 chromatography step as described for the head domain constructs
except that induction with
isopropyl-1-
Cells containing induced GSTase fusion proteins were lyzed and
clarified essentially as described for DKH340 except that the buffer
was 50 mM Bicine/NaOH, pH 8, with 10 mM EDTA.
The clarified extract was applied to a glutathione-Sepharose column,
washed with 20 mM Bicine/NaOH, pH 8, containing 1000 mM NaCl and 2 mM EDTA. The protein was eluted
with 20 mM Bicine/NaOH, pH 8, containing 50 mM
NaCl, 2 mM EDTA, and 5 mM glutathione. Control
GSTase was obtained by expression of pGEX-2T (Pharmacia).
Inclusion Bodies--
When the long non-fusion constructs
(DKH894-DKH975) are induced at 37 °C, the pool of soluble construct
is vanishingly small and essentially all of the expressed protein is
present as inclusion bodies. For isolation of inclusion bodies, cells
that had been induced at 37 °C were lysed and centrifuged as
described above. The pellet was washed by resuspension and
centrifugation at 10,000 × g successively in MgM
buffer (14) with 0.5 M NaCl, 0.5 M NaCl and 1%
Triton (twice), and finally with 0.05 M NaCl (twice). The washed pellets were solubilized by heating to 100 °C in SDS-PAGE sample buffer and used as size markers for unproteolyzed constructs.
Proteolysis--
Constructs containing the extreme COOH terminus
are highly susceptible to proteolysis during isolation and the
particular preparations used here have some proteolytic cleavage as
indicated in Fig. 1. For the GSTase fusions, the largest species is the size expected for a full-length construct in each case (Fig.
1A). The proteolytic cleavages are not likely to have
occurred in the GSTase domain because GSTase itself is resistant to
proteolysis and because all of the proteolytic fragments continue to
bind tightly to glutathione-Sepharose. Also, in preparations that have been more extensively proteolyzed, the ladder of fragments shifts down
toward the mass for GSTase, but not below, as expected for proteolytic
removal of the kinesin-derived COOH-terminal region. The longer
fragment (PF-I) results from cleavage at
Full-length DKH975 is also partially proteolyzed to species
corresponding to cleavage at the PF-I and PF-II sites as indicated in
Fig. 1B. Proteins obtained from inclusion bodies were used for size standards as they have likely precipitated before undergoing proteolysis. Although there is some unproteolyzed protein, the major
band in a typical preparation of DKH975, as indicated in Fig.
1B, corresponds to cleavage at
the PF-I site. There is also a significant band corresponding to
cleavage at the PF-II site. Some preparations of DKH975 are more highly
proteolyzed with the major band corresponding to PF-II and these
preparations have not been included in the work presented here.
Preparations of DKH960 show much less proteolysis and appear
predominately as a single band of the expected size. Although it cannot
be excluded that some proteolysis of DKH960 has occurred to produce the
PF-I product, which is approximately the same size as DKH960, there is
no major accumulation of the PF-II product as observed with DKH975. All
of the shorter constructs (DKH894-DKH945) migrated as single bands at
the same position as the corresponding proteins isolated from inclusion
bodies.
Salt Dependence of Sedimentation of Long Heavy Chain
Constructs--
The sedimentation coefficients of the full-length
Drosophila kinesin heavy chain construct, DKH975, and
truncated DKH960 are 5.4 S in A25 buffer with 25 mM KCl and
1 M NaCl, but shift to 6.7-7.0 S at low salt concentration
with a midpoint for the transition of ~0.5 M NaCl (Fig.
2A). The behavior of these
long Drosophila constructs is essentially identical to that
of the bovine kinesin heavy chain dimer (11). The elution position of
DKH975 and DKH960 during gel filtration on Bio-Gel A-5m at high and low
salt (not shown), as well, is essentially identical to that of the
bovine heavy chain dimer (11) with an increase in Stokes radius at high
salt. The similarity of the sedimentation and diffusion properties to
those of bovine dimer indicates that DKH975 and DKH960 are also dimers
that undergo a transition from a compact conformation at low salt to an
extended, highly asymmetric, conformation at high salt concentration. A
possible model for this transition is presented in Fig. 2B
as a framework for discussion.
The sedimentation coefficient of DKH894, DKH910, and DKH927 remain
In the preparations of DKH975 that were used, the major component was a
proteolysis fragment, PF-I, of approximately the same size as DKH960
(see "Materials and Methods"). Thus the observed sedimentation
behavior of DKH975 preparations is dominated by the PF-I species and
does not necessarily represent the behavior of true full-length DKH975.
At 300 mM NaCl, DKH960 should be in the compact
conformation, but DKH945 and shorter should be mainly extended.
Chromatography of DKH975 preparations at 300 mM NaCl indicates that full-length DKH975 comigrates with the PF-I species at
the position expected for the compact conformer, and thus it is likely
that full-length DKH975 does behave similarly to DKH960. The smaller
PF-II species elutes earlier at the position expected for the extended
conformer, consistent with its length of ~940.
Localization of the Site of Interaction in the
NH2-terminal Region--
The inability of DKH894-DKH927 to
from the compact conformer at low ionic strength suggests that
formation of the compact conformer results from interaction of the
COOH-terminal region with regions closer to the head domain. A GSTase
fusion protein containing the kinesin heavy chain from amino acids
864-975, designated GST864-975, was used to determine if it could
interact with head domains in a salt-dependent manner.
GST864-975 was loaded onto a column containing glutathione-Sepharose
and head domains were passed down the column in low salt buffer to
determine if binding could occur. As indicated in Fig.
3A, DKH365, DKH381, and DKH392 were retained by the column, whereas DKH340 and DKH357 passed through.
Subsequent elution of the columns with high salt buffer released the
bound DKH365, DKH381, and DKH392.
Localization of the Site of Interaction in the COOH-terminal
Region--
In order to further localize the site of interaction in
the tail region, GSTase fusions containing smaller parts of the
COOH-terminal region were tested for interaction with DKH392. As
indicated in Fig. 3B, the region between 893 and 975 was
sufficient for salt-dependent interaction, but the region
between 864 and 893 was not sufficient for tight interaction. Column
binding tests were also performed with GSTase fusions containing
kinesin tail sequences from 893 to 937 and 893 to 960 as indicated in
Fig. 4. Under these conditions, none of
the three head domains binds to a GSTase control, GST893-910 (not
shown), or GST893-937. Extension of the tail domain to 960 (GST893-960) does produce salt-dependent binding of DKH365
and DKH405, but not DKH346. However, the binding of DKH405 is stronger than that of DKH365, as DKH365 is rapidly eluted by 150 mM
salt, whereas higher concentrations of salt are required to rapidly elute DKH405.
Comigration of Head and Tail Regions during Centrifugation at Low
Salt Concentration--
Sucrose density gradient centrifugation of
DKH392 alone in the absence of added salt (Fig.
5A) gives a sedimentation
coefficient of 5.9 S compared with 5.2 S observed previously in 25 mM KCl (22). The self-aggregation of DKH392 is more
extensive at lower salt (3) and this shift in
s20,w value likely results from some
reversible aggregation under these conditions. Sedimentation of
GST893-975 alone gives a sedimentation coefficient of 4.4 S that is
consistent with a dimeric protein of its subunit molecular weight and
is similar to the value of 4.4 S obtained for the parent dimeric GSTase
(not shown). The preparation of GST893-975 used here is more
extensively proteolyzed than the sample of GST893-975 in Fig.
1A. The proteolytic fragment PF-II is the most abundant species and only a minor amount of PF-I is present, but considerable full-length GST893-975 is still present.
When GST893-975 is mixed with an excess of DKH392 before
sedimentation, full-length GST893-975 comigrates with DKH392 at a higher sedimentation coefficient of ~7 S (Fig. 4C). Most
of the major proteolytic fragment (PF-II) of GST893-975 does not
migrate more rapidly in the presence of DKH392, but some of the
proteolytic fragment does shift as indicated by streaking into
fractions further down the gradient. Weaker interaction of PF-II with
DKH392 is consistent with the size of the COOH-terminal truncation to
~940 (Fig. 1B) and the shift in the midpoint for DKH937
and DKH945 that is observed in Fig. 2. The parent GSTase protein shows
no shift in sedimentation coefficient when centrifuged under these conditions in the presence of excess DKH392 (not shown).
The interaction observed between GST893-975 and DKH392 is likely to be
weak and reversible under these conditions and is only observed in Fig.
5C because DKH392 was in excess. Sedimentation of
GST893-975 with excess DKH392 in buffer with 25 mM KCl
results in only a partial shift of GST893-975 (not shown), indicating that even low concentrations of added salt are sufficient to inhibit association. GST893-975 also fails to comigrate with DKH392 in the
absence of added salt when the concentration of DKH392 is reduced (not
shown), indicative of a weak interaction that requires high levels of
DKH392 to maintain a significant fraction of the GST893-975 as the complex.
ATPase Rates--
The initial rates for ATP hydrolysis was
determined for compact DKH975 and extended DKH894 in A25 buffer with 50 mM KCl (Fig. 6). The
kcat and K0.5(MT)
values were 35 and 3.8 s The heavy chain dimer of bovine kinesin has previously been shown
to undergo a transition from an extended conformer at high salt
concentration to a more compact conformer at low salt concentration (11). The bovine dimer, however, likely results from proteolysis of the
light chains during isolation of the native bovine heterotetramer (11,
23) and the heavy chain dimer could potentially still contain small
light chain domains that are not easily detected by SDS-PAGE. The
ability of the Drosophila heavy chain dimer obtained by
expression in E. coli to form the compact conformer at low ionic strength indicates that the heavy chain itself possesses the
capability to form a compact conformation without the involvement of
light chains or eukaryotic post translational modification.
Two types of mechanisms could produce the compact conformer. One
possibility is that the stalk is bent back on itself at the likely
hinge at position ~600 and that the interaction of the front and back
half of the stalk along their lengths provides the major stabilization
of the compact conformer. In the other type of model, as indicated in
Fig. 2B, formation of the compact conformer is driven by a
favorable interaction between a domain in the NH2-terminal
head region and a domain in the COOH-terminal tail region that can
occur because there is sufficient flexibility in the stalk for the two
ends of kinesin to come into contact. The break in the predicted
coiled-coil of the stalk around position 600 is a candidate hinge for
either model. The inability of DKH927 to form the compact conformer at
low salt (Fig. 2A) indicates that the COOH-terminal region
between 927 and 960 is required and, conversely, that the stalk domain
itself cannot produce a stable compact conformation. This suggests that
the compact conformer is not produced by interactions within the stalk,
but rather by interaction between specific segments in the
NH2- and COOH-terminal regions as indicated in Fig.
2B. Such a direct interaction is supported by the
observation that separately expressed tails and heads interact in a
salt-dependent manner (Figs. 3-5). This does not totally
preclude involvement of antiparallel interactions between the front and
back half of the stalk domain in the compact conformer, but does
indicate that any such interactions within the stalk are not the major
energetic driving force for formation of the compact conformer.
Analysis of the tendency for coiled-coil formation indicates (Fig.
7) that the region between 850 and 910 is
likely to be coiled-coil in heptad frame 7N with a weak area
around Pro883. The region between 910 and 930 has a weaker
predicted tendency for coiled-coil formation in heptad frame
7N-3. Detailed inspection (Fig. 7) indicates that there is a
reasonable heptad repeat in both heptad frames 7N-3 and
7N, that could facilitate a 4-helix or antiparallel
coiled-coil interaction between the ~883 and 930 region of the tail
and the helical region of the neck.
1 and 35 s
1, respectively, for kcat at
saturating microtubules).
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1 at saturating MT concentration that is
observed with dimeric head constructs (see Ref. 3) is consistent with
tight coupling of 1 ATP per step during motility (40 s
1
per head × 2 heads per dimer × 8 nm per tubulin dimer along
protofilament predicts a sliding rate of ~600 nm/s that is equal to
the observed sliding rate of single molecules, see Ref. 14, for
discussion). The maximum ATPase rate of bovine kinesin in the compact
conformer, however, is only ~1 s
1 for the 9 S
heterotetramer and ~7 s
1 for the 7 S dimer (15) and
this suggests that the inhibition of full-length kinesin
versus free head domains is due to interaction of the heads
with tail domains in the compact conformer. Myosin from smooth muscle
and nonmuscle cells undergoes a similar conformational transition at
low salt concentration that produces a compact form with a highly
inhibited rate of actin-stimulated ATP hydrolysis (16, 17).
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-D-galactopyranoside was performed at
25 °C. MgATP was included at
0.1 mM in all
solutions. Solid ammonium sulfate was added to the pooled P-11
fractions to 36% and centrifuged to collect the kinesin in the pellet.
Both bovine kinesin and long heavy chain constructs of
Drosophila kinesin precipitate at this low concentration of
ammonium sulfate. Shorter Drosophila kinesin constructs, as
well as most Escherichia coli proteins, remain soluble and
this step provides considerable further purification. After dissolving
the ammonium sulfate pellet in minimal A25 with 1 M NaCl,
it was subjected to gel filtration on Bio-Gel A-5m in 1 M
NaCl. The peak fractions were dialyzed versus KA buffer
(14), loaded on a Bio-Gel DEAE column, and eluted with 150 mM NaCl in KA buffer. The pooled fractions were dialyzed
against A25 buffer with 50 mM KCl in 50% glycerol with 2 mM dithiothreitol and stored in small aliquots at
80 °C.
960 as this fragment of
GST893-975 comigrates slightly faster than uncleaved GST893-960. The
short fragment (PF-II) results from cleavage at ~940 as this fragment
of GST893-975 and GST893-960 is slightly larger than GST893-937. In
more extensively proteolyzed preparations such as that used in Fig. 5,
the fragment terminating at ~940 is the major species. Constructs
lacking the 940-975 region are not significantly proteolyzed.
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Fig. 1.
SDS-PAGE analysis of protein
preparations. A, SDS-PAGE of GSTase tail fusion
proteins. M, molecular weight standards; G,
GSTase; A, GST864-975; B, GST893-975;
C, GST893-960; D, GST893-937 and E,
GST893-910. Arrows indicate position for GST864-975 of the
two major proteolytic fragments (PF-I and PF-II) formed by cleavage of
the extreme COOH-terminal region. B, SDS-PAGE of non-fusion
constructs. Proteins from inclusion bodies are indicated as
Ixxx, where xxx is the length of the construct.
Only the region corresponding to the full-length species is shown. The
long constructs differ only slightly in relative size and tall gels
(Hoefer SE280) with 6% acrylamide were required for even partial
separation. Also the time for electrophoresis was increased past the
time required to elute the tracking dye so that the long constructs
migrated closer to the bottom of the gel. The proteins were loaded at a
low level that was just detectable by sensitive staining with colloidal
Coomassie G-250 (31). Even moderate increases in loading produced bands
that were too wide for resolution and migrated faster as illustrated by
the two loadings of DKH960 at the right of the gel.
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Fig. 2.
A, dependence of
s20,w on salt concentration for heavy
chain dimers. Sucrose density centrifugation was performed in A25
buffer with 0.05-0.1 mM MgATP. The values at the lowest
ionic strength are for A25 buffer alone. Higher ionic strengths were
obtained by supplementing A25 buffer with 25 mM KCl and
0-1 M NaCl. Variable aggregation to species larger than
the dimer was observed at low ionic strength and high concentration,
especially for Lys927. The concentration dependence of all
of the constructs (except for DKH975) was investigated at low ionic
strength and the reported s20,w values
at less than 100 mM ionic strength were obtained at very
low initial protein concentration (<0.02 mg/ml) where aggregation was
not a problem. DKH975, solid circles; DKH960, open
circles; DKH945, squares; DKH937, open
diamonds; DKH927, open triangles; DKH910, filled
triangles; and DKH894, filled diamonds. B,
model for conformational transition. The shaded ovals
represent head domains and the smaller solid ovals represent
the COOH-terminal non-helical domains. The specific interaction that is
indicated for the compact 7 S conformation has part of the
COOH-terminal non-helical domain interacting with the motor domain and
the adjacent potentially helical region of the tail interacting with
the coiled-coil neck. This detailed interpretation is speculative, but
is consistent with the localization of the regions that are required
for formation of the compact conformer as developed under
"Discussion."
5.6 S (Fig. 2A), even at low salt and thus removal of the
COOH-terminal 48 amino acids inhibits formation of the compact conformer. Deletions of intermediate length, DKH945 and DKH937, still
form the compact conformation at low salt, but the midpoint for the
transition is shifted to lower salt concentration. The midpoints for
DKH945 and DKH937 are similar, but DKH937 is shifted slightly to lower concentrations.
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Fig. 3.
Binding of head domains to GSTase tails.
Columns of glutathione-Sepharose (0.2-ml bed volume) were loaded with
GSTase fusion proteins (0.3 mg) and washed with A25 buffer containing 1 M NaCl to remove unbound fusion protein. Following
equilibration with A25 buffer containing 0.05 mM MgADP,
head constructs were loaded (0.1 ml of 0.13 mg/ml) and washed with a
total of 0.8 ml of the same buffer. The combined eluents were pooled as
the low salt run-through fraction. The columns were then washed with
0.35 ml of additional A25 and eluted with 0.6 ml of A25 with 1 M NaCl as the high salt elution fraction. The low salt run
through fraction and the 1 M NaCl elution fractions were
analyzed by SDS-PAGE as shown. Recovery of the head constructs in the
combination of the low and high salt fractions was essentially
complete. A, binding of different head constructs to columns
containing bound GST864-975. B, binding of DKH392 to
columns containing different GSTase tail fusions or control
GSTase.
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Fig. 4.
Binding of heads to GST893-937 and
GST893-960. Columns of glutathione-Sepharose (1 ml bed volume)
were loaded with GSTase fusion proteins (2.5 mg). A mixture of DKH346,
DKH365, and DKH405 was loaded on the columns (1 ml of 0.2 mg/ml each)
in A25 buffer with 10 mM NaCl and washed with a total of
1.5 ml of buffer with 10 mM NaCl. The combined eluents were
pooled as the run-through fraction (RT). The column was then
washed sequentially with increasing salt concentrations of 10, 150, and
1000 mM NaCl (2.5 ml total for each). Fractions were
analyzed by SDS-PAGE. Loading of each fraction was adjusted so that the
protein concentration would equal that of the preload if the recovery
was 100% in that fraction.
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Fig. 5.
Gradient centrifugation of mixture of DKH392
and GST893-975. SDS-PAGE of fractions from sucrose gradient
centrifugation of DKH392 and GST893-975 with standard proteins in A25
buffer without added salt. Centrifugation was for 18 h at 4 °C
in an SW41 rotor (Beckman) at 40,000 rpm. A, DKH392;
B, GST893-975; and C, both DKH392 and
GST893-975. BSA, bovine serum albumin; C.A.,
carbonic anhydrase; 893, full-length GST893-975;
P.F., proteolytic fragment PF-II of GST893-975; and
392, DKH392.
1 and 1.9 and 0.27 µM, respectively, for DKH894 and DKH975.
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Fig. 6.
MT-stimulated ATPase. The MT-stimulated
ATPase was determined for 30 nM kinesin heavy chain in A25
buffer with 50 mM KCl, 1 mM MgATP, 2 mM potassium phosphoenolpyruvate, and 0.3 mM
NADH with pyruvate kinase and lactate dehydrogenase.
Squares, DKH894; diamonds, DKH975. The reactions
were not stirred after initial mixing.
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Fig. 7.
Analysis of tail sequences that are required
for formation of the compact conformation. A, results
of a multiple sequence alignment performed by ClustalW (MacVector,
Oxford Molecular Group) on conventional kinesins (Drosophila
(32), human (33), human neuronal (34), mouse (GenBank, X61435), squid
(35), and urchin (36)). The indicated sequence is that for
Drosophila with a "*," indicating absolute conservation
in all 5 species and a ":," indicating only conservative changes.
The strong conservation observed at the beginning of this region
extends into the area on the NH2-terminal side of position
881, but there is no strong conservation on the COOH-terminal side of
position 950. Positively and negatively charged residues are indicated
by + and , respectively, on the line above the sequence.
The heptad repeat positions a and d are also
indicated above the sequence for heptad frames 7N-3 and
7N. B, prediction of coiled-coil tendency for
Drosophila kinesin heavy chain determined by the
program MacStripe2.0 ((37),
http://www.york.ac.uk/depts/biol/units/coils/coilcoil.html)
implementing the COILS2 algorithm of Lupas (38). Solid line
is for a window size of 28 residues with a shift in predicted heptad
frame from 7N to 7N-3 at position 912. The
dashed line is for the more stringent case of a window size
of 21 residues and it emphasizes the weaker strength of the prediction
for the region around Pro883 and for the region between 912 and 930. The arrow indicates the end of the
Drosophila sequence at 975.
The ability of DKH937 to form the compact conformer at low salt (Fig. 2A) and the ability of GST893-960 to bind to DKH365 (Fig. 4) indicate that the tail region from 893-937 and the head region of 1-365 are sufficient for tight interaction, although interaction of additional regions may provide further stabilization. The NH2-terminal half of the coiled-coil neck is likely to play a critical role as even partial deletion (DKH357 versus DKH365) strongly inhibits interaction with the tail domains. These regions of both the neck and tail are highly charged (16 charged for 340-365 and 15 charged for 912-936) and this could account for the high salt dependence of the interaction. The whole region between 883-936, however, has an abundance of charged groups and is it not possible to definitely predict the alignment of the tail and neck regions in the complex. Although the NH2-terminal half of the neck is likely to be important for the binding of the tail domain, several constructs containing the neck and adjacent regions, in the absence of complete head domains, have been tested for binding to the tail domain and none have been found to bind strongly.2 It is thus likely that strong binding to the tail domain requires interaction over a more extended region than just the NH2-terminal half of the coiled-coil neck. Although the ability of DKH937 to form a compact conformation at low salt indicates that the region beyond 937 is not absolutely required, the shift in the midpoint for the transition with longer constructs indicates that the region beyond 937 can stabilize the interaction. If the region of ~345-365 interacts with ~910-930 in an antiparallel configuration as in Fig. 2B, then the region of the tail beyond 930 would be juxtaposed to the head domain and in a position for potential interaction. Conventional kinesins show strong sequence conservation in this region up to position 950 (Fig. 7) and the conserved region (940-950) surrounding the IAK at positions 942-944 is thus a candidate for this additional area of interaction with the head. This antiparallel alignment would also juxtapose the COOH-terminal half of the neck with the region between ~883 and 910 and interaction of these regions could be responsible for the stronger interaction of DKH405 versus DKH365 with tails (Fig. 4).
The highly charged NH2-terminal half of the coiled-coil neck has a gap in the hydrophobic heptad repeat and this region was postulated to be capable of reversibly uncoiling, while the dimer remained anchored by the stronger interactions in the better heptad repeats of the COOH-terminal half of the neck (24). Recent work with peptides from the neck region has established that such uncoiling of the NH2-terminal half of the neck can occur (4, 5). Although the exact oligomeric state of the complex between the tail and head/neck is not known, the interaction between monomeric DKH365 and GST893-975 suggests that formation of a 4-helix bundle is not necessary, at least for the NH2-terminal half of the neck. An alternative possibility is that strand displacement may occur with uncoiling of the NH2-terminal half of the neck to allow formation of a pair of antiparallel coiled-coils with the region from ~910 to 930 of the tail. The relatively poor prediction for a parallel coiled-coil interaction of this region of the tail with itself may be reflective of an actual role in antiparallel interaction with the neck.
Fungal kinesins are relatives of conventional kinesin (25) that have lost the highly charged nature of both the neck and tail regions that are homologous to those implicated above in formation of the compact conformation. Either these regions of fungal kinesins do not interact, or both regions have co-evolved to maintain an interaction that is not as highly dependent on charge. Interestingly, the IAK region is, however, conserved between conventional and fungal kinesins.
Entropic effects are likely to be the major cause of the weaker apparent interaction of domains on separate constructs relative to domains that are covalently linked. For example, DKH975 remains predominantly in the compact conformation at 0.3 M salt, whereas interaction of GST893-975 and DKH392 during cosedimentation is weak and readily reversible even in the absence of added salt. When the domains are on separate constructs, they are free to diffuse away from each other following dissociation, whereas the dissociated domains of DKH975 are restrained to remain near each other because they are covalently linked through the stalk. The failure of DKH405 or DKH365 to bind to GST893-937 in the column binding assay (Fig. 4) while DKH937 can still form the compact conformer, likely also reflects the more stringent requirements for association of fragments that are not covalently linked. Additive contributions from the entire extended binding region including the IAK region that is present in GST893-960 are apparently required for binding to be tight enough to be observed under these more stringent assay conditions. Entropic considerations also predict that the column binding experiments (Figs. 3 and 4) can better detect weak interactions compared with cosedimentation experiments (Fig. 5). The sedimentation velocity of an equilibrating mixture is directly related to the fraction associated; but in the column binding assay, head domains must interact sequentially with many bound GST-tail fusions before they can be eluted.
The analysis of ATPase rates of heavy chain constructs that are close
to full-length is complicated by the tendency for formation of asters
of MTs in the presence of ATP as originally reported by Urrutia
et al. (26). The heavy chain constructs used here can also
produce such asters during the course of an ATPase assay at high
concentration as verified by electron
microscopy.3 The ATPase
results in Fig. 6 were obtained as initial rates at low kinesin
concentration (30 nM) in order to minimize the contribution from kinesin molecules in asters. Extended DKH894 has a high
kcat at saturating MT concentration that is
similar to that of fully active shorter constructs (3). The
kcat of DKH975, however, is 10-fold lower,
indicating inhibition in the compact conformer. The true rate of DKH975
in the compact conformer may be even lower than the observed rate of
3.4 s1 as the K0.5(MT) of
0.27 µM is unexpectedly low and part of the observed
activity likely represents catalysis by the contaminating PF-II species
which has a high ATPase rate and a low
K0.5(MT) value.2 With bovine kinesin,
inhibition of MT-stimulated ATPase activity results from inhibition of
the ability of MTs to stimulate ADP release from the compact conformer
that is present at low ionic strength (15). This inhibition could
result directly from steric blockage by the tail of the MT interaction
site on the head or indirectly by a conformational change in the head
or neck that is induced by binding of the tail domains.
If the site on the tail domain to which the head domain bound was
overlapping with the site on the tail to which the vesicle receptor
bound, then competition between these two binding modes would provide a
natural basis for regulation by mass action (11) as illustrated by the
model of Fig. 8. Even if the
receptor-binding site is on the light chains, the binding of tails to
heads versus receptors could be still be competitive as long
as steric constraints prevented both interactions from occurring at the
same time. When free in solution, kinesin would be in the compact
conformer and be inhibited, while binding of the membrane receptor to
the tail would release the heads in their active form. If the binding
of the tail to the receptor was stronger than binding of the tail to
the heads and if kinesin was in excess, then all the receptors would be
occupied with active kinesin and any excess kinesin would be in the
compact conformer and thus inhibited. Such binding-induced activation
of kinesin has been reported with nonphysiolgical surfaces (27-29) and
is likely to also be responsible for the high motor activity of
adsorbed kinesin in motility assays, but activation by physiological
receptors remains to be demonstrated. The occurrence of
receptor-induced activation by induction of the extended conformer would not preclude additional or alternative regulation by other means
such as phosphorylation (30).
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ACKNOWLEDGEMENTS |
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We thank the Drug Synthesis and Chemistry Branch of the National Cancer Institute for Taxol, L. S. B. Goldstein for providing Drosophila kinesin cDNA, and S. Admarral and J. Negy for assistance in cloning and preliminary experiments.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NS28562 and National Science Foundation Grant REU DBI-9424036 (to C. E. and B. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biological Sciences, University of
Pittsburgh, Pittsburgh, PA 15260.
§ Present address: Dept. of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260.
¶ To whom correspondence should ba addressed: Dept. of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-268-3244; Fax: 412-268-7129; E-mail: ddh+{at}andrew.cmu.edu.
2 M. Stock and D. Hackney, unpublished observations.
3 D. Hackney, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: MT, microtubule; PAGE, polyacrylamide gel electrophoresis; Bicine, N,N-bis(2-hydroxyethyl)glycine; GST, glutathione S-transferase.
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REFERENCES |
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