(Received for publication, October 3, 1994; and in revised form, April 7, 1995)
From the
Evidence is presented that MgADP can be noncovalently trapped in
myosin subfragment 1 in the presence of ScF resulting in the concomitant loss of ATPase function. The
rate of inactivation in the presence of MgCl
at 25 °C
is 8.7 M
s
which is too
slow for a simple collisional mechanism and suggests that a subsequent
slow isomerization step is responsible for formation of a stable
ternary complex, S1
MgADP
ScF
in a
manner analogous to that proposed for the Vi stabilized complex by
Goodno (Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2620-2624). It is also found that ADP can be trapped in
subfragment 1 in the absence of MgCl
indicating the
formation of an S1
ADP
ScF
complex. The
stability of these complexes at 4 °C was studied by following the
loss of trapped [
C]ADP with a chase with ADP.
The rate of nucleotide loss at 4 °C was biphasic for both complexes
suggesting that the inhibitory complexes exist in two distinct states
as previously proposed for the ternary complex stabilized by Vi
(Mihashi, K., Ooi, A., and Hiratsuka, T.(1990) J. Biochem. (Tokyo) 107, 464-469). Formation of these complexes resulted in a
marked enhancement of the intrinsic tryptophyl fluorescence suggesting
that conformationally they may resemble the steady-state intermediate
formed with MgATP. The failure to observe photolysis in the presence of
excess Vi at sites associated with the ATP consensus sequence suggests
that in these complexes ScF
occupies the site
responsible for these cleavage reactions and that it is not displaced
by the added Vi.
The energy for muscle contraction is derived from the hydrolysis
of MgATP by myosin and the activation of this process by actin. During
this process, myosin forms a series of intermediates resulting in the
formation of a steady-state intermediate MADPP
which
upon reassociation with actin forms AMADP
P
, a
non-force-producing state, which subsequently isomerizes to
force-bearing states with and without P
(AMADP
P
or AMADP), respectively (Hibberd and Trentham, D. R.(1986);
Dantzig et al.(1992)). Thus, a key question related to the
structural basis of force generation is how different is the S1 (
)structure in the MADP
P
and
MADP
P
or MADP states. Unfortunately, the former two
intermediates have only limited lifetimes, and it appears that the
latter state when bound to actin is different in structure from that
formed by the binding of MgADP to actoS1 (Sleep and Hutton, 1980).
Therefore, stable analogs corresponding to these species would be
useful in obtaining information of their structures.
Since the
demonstration by Goodno(1979) that orthovanadate ion in the presence of
MgADP was able to form a stable ATPase-inhibitory complex with myosin
S1, corresponding to a 1:1:1 ternary complex designated MADPVi,
much interest has been devoted in its characterization as it appears to
have some properties in common with the steady-state intermediate,
MADP
P
, of the MgATPase hydrolysis cycle by S1 (Goodno
and Taylor, 1982; Smith and Eisenberg, 1990; see also Goodno(1982) for
a review)). Because of the simple stoichiometry of the complex and
because of its inhibitory nature, it is generally assumed that the
nucleoside diphosphate resides at the nucleotide binding site with the
Vi taking the place of the terminal
P
group and, in
this way, forms a stable analog of the transition state. Such an
interpretation is also supported by the fact that structurally the
vanadate ion is similar to orthophosphate (Lindquist et al.,
1973).
More recently, the use of other complex anions with
similarity to P has been investigated for their ability to
stabilize the transition states of phosphotransferases and
nucleotidases in the presence of nucleotide diphosphate (Chabre, 1990).
These include fluoride complexes of beryllium and aluminum,
BeF
and AlF
, respectively,
and their efficacy in forming stable transition state analog states has
been studied with actin (Combeau and Carlier, 1989, 1990), with smooth
muscle heavy meromyosin (Maruta et al., 1993) and skeletal
myosin S1 (Phan and Reisler, 1992; Beck et al., 1992: Werber et al., 1992; Henry et al., 1993).
In the present
paper we present evidence that fluoroscandium anion (presumably
ScF) can also lead to effective trapping of ADP in
S1 in both the presence and absence of Mg
with
concomitant inactivation of the ATPase properties of the protein.
Surprisingly, free nucleoside diphosphate is more stably trapped than
the MgADP form with fluoroscandium. Actin binding to these complexes
results in a faster release of trapped ADP in accord with the usual
effect of actin in accelerating product release from S1.
Distilled water further purified through a Millipore QTM
system was used throughout. ATP, ADP, dithioerythritol, HEPES, Tris,
NaF, KF, sodium orthovanadate, BeCl, AlCl
, and
chymotrypsin were from Sigma and Aldrich. ScCl
was
purchased from Johnson Mathey Electronics with a purity of 99.9%. A
stock solution of ScCl
(100 mM) was made in water
and stored at -20 °C. [
C]ADP (specific
activity 2.4
10
cpm/mol) was purchased from DuPont
NEN. All other reagents were of analytical grade.
Incubation of S1 at 25 °C for 30 min in the presence of
0.2 mM ADP, 0.5 mM ScCl, and 10 mM NaF in buffer A resulted in virtually complete inhibition of the
EDTA ATPase of the protein. ADP and NaF were found to be essential for
this inhibitory action since omission of either one had no effect on
the ATPase activity of the S1. However, the presence of Mg
ions was not required to produce inhibition with ADP and
fluoroscandium, except in this case lower extents of inhibition were
observed at low concentrations of scandium (Fig. 1). The
inactivation was studied as a function of the Sc
concentration, and the data are shown in Fig. 1in the
presence of ADP (no MgCl
) and MgADP. In the latter case,
90% inhibition was observed with 0.1 mM scandium. The
concentration dependence of the inhibition in the absence of
Mg
ions is more complex and 90% inhibition required
about 0.2 mM scandium. The loss of ATPase function is
consistent with the formation of a stable inhibitory complex, and,
since ADP, scandium, and NaF are all essential for this to occur, it is
likely that this complex involves formation of a ternary complex in
which MgADP or ADP and ScF
are simultaneously
bound to the S1.
Figure 1:
Effect of
concentration of scandium chloride (), beryllium chloride
(
), and vanadate (
) on the formation of an inhibitory
complex with S1 in the presence of ADP. S1 (17.4 µM) was
incubated at 25 °C (in the presence or absence of 1.0 mM MgCl
) containing 0.03 M Tris, 10 mM NaF (or KF for trapping with beryllium and scandium), and 0.2
mM ADP at pH 7.9 (adjusted at room temperature). After 5 min,
the P
analogs were added (up to 200 µM) and
incubated for an additional 30 min. Aliquots were then removed and
assayed for EDTA/K
ATPase activity as described under
``Materials and Methods.''
, scandium chloride in the
absence of MgCl
.
The presence of MgATP in the incubation solution
retards the onset of inhibition as shown in Fig. 2. This is
consistent with the S1 being primarily in the MADPP
conformation and the occupancy of the site with MgADP and P
with the latter retarding the binding of
ScF
. The rate constant (k
)
for the inactivation in the presence of 500 µM scandium
was obtained from the semilogarithmic plot of the residual ATPase
activity versus time and has the value of 2.8
10
s
, corresponding to an
apparent second order rate constant (k
) of 0.056 M
s
. The latter second
order rate constant is about an order of magnitude lower than that
observed with Vi and beryllium suggesting weaker binding of the
ScF
to S1
MgADP (Goodno, 1979; Phan and
Reisler, 1992).
Figure 2:
Effect of the presence of MgATP (1.0
mM) on the rate of formation of P by S1 in the
absence and presence of ScF
. To S1 (0.5
µM) at 25 °C in the presence of 5.0 mM MgCl
, 1 mM ADP, 0.03 M HEPES, and 10
mM NaF at pH 7.0 (adjusted at room temperature) was added
MgATP (1.0 mM), and the rate of P
formation was
followed in the absence (
) and presence (
) of ScCl
(500 µM).
The inactivation of the ATPase activity induced by
preincubation of S1 with MgADP in the presence of ScCl and
KF was followed as a function of the preincubation time, and the data
are shown in Fig. 3. The rate constant (k
) for the inactivation in this case was
obtained from the slope of the semilogarithmic plot of the residual
activity versus time and was found to be 1.73
10
s
yielding an apparent second
order rate constant of 8.7 M
s
. While clearly faster than the apparent
second order rate constant measured for the inactivation in the
presence of MgATP, both rates are far too slow to correspond to a
simple collisional process. It is more likely that the inactivation
proceeds in a manner analogous to that suggested for Vi by Goodno(1979)
and involves a rapid equilibrium binding step of ScF
to S1
MgADP followed by a slow isomerization step.
Analyses of the amount of scandium trapped in the two complexes were
done in triplicate on two different preparations of S1 and yielded 1.78
± 0.3 and 1.61 ± 0.2 mol of scandium per mol of S1 for
the inhibitory complexes containing MgADP and ADP, respectively. Since
the amounts of nucleotide trapped in the S1 by the fluoroscandium anion
were 0.72 ± 0.2 and 0.70 ± 0.1 for MgADP and ADP,
respectively, per mol of S1, it appears that close to 2.0 mol of
scandium per mol of S1 are present in these complexes. The amount of
magnesium present in the complex formed with and without MgCl
was 0.85 ± 0.02 and less than 0.14, respectively. S1
incubated with ScF
in the absence of MgCl
and ADP showed less than 0.14 mol of scandium per mol of S1 after
gel filtration centrifugation indicating that no high affinity binding
site exists for ScF
when ADP is absent. Assuming
that the total inactivation of the S1 ATPase is associated with the
binding of 2.0 mol of Sc
, a Scatchard analysis (data
not shown) of the inactivation data versus free ScF
gave an association constant of approximately 6.0
10
M
. This is about an order of
magnitude lower than that reported for the binding of BeF
to S1
MgADP (Phan and Reisler, 1992).
Figure 3:
Time dependence of inactivation of S1 by
ScF. S1 was incubated as described in Fig. 1(10 mM KF for beryllium and scandium), and the
inactivation was followed as a function of time upon addition of
ScCl
(
), BeCl
(
), or Vi (
) to
a final concentration of 200 µM as described under
``Materials and Methods.''
, inactivation by ScCl
in the absence of MgCl
.
The stability
of the inhibitory complexes formed in the presence or absence of
MgCl was next examined by following the release of bound
radiolabeled ADP by a chase experiment with 1.0 mM ADP. The
results are shown in Fig. 4for the ternary complexes formed
with either ADP or MgADP. The data for both processes indicate that the
release is biphasic, with the fast phase having the same apparent rate
constant (k
) for both complexes of 8.0
10
s
. However, the fractions of
fast and slow components are quite different. In the case of the
MgADP-containing complex, the fast component corresponds to about 0.46
of the total S1, while for the ADP-containing complex the fast fraction
corresponds to only about 0.11 of the total S1. The slower phases
yielded apparent rate constants of 8.0
10
s
and 3.9
10
s
, corresponding to half-lives of 24 and 49 h
for the MgADP- and ADP-containing inhibitory complexes, respectively,
at 4 °C.
Figure 4:
Rate of trapped
[C]ADP release. The inhibitory complexes formed
with [
C]ADP and ScF
in the
presence (
and absence (
) of MgCl
were isolated
at 4 °C, and MgADP and ADP were added immediately to a final
concentration of 1.0 mM respectively. The release of
[
C]ADP was followed as described under
``Materials and Methods.''
Actin is known to be able to decompose the ternary
inhibitory complexes formed between S1 and MgADP with Vi (Goodno and
Taylor, 1982) or BeF or AlF
(Phan and Reisler, 1992; Werber et al., 1992). The
decomposition of the inhibitory complex is conveniently followed by the
regain of the EDTA ATPase activity which is measured at high ionic
strength where the activating effect by actin is minimal. The results
of such an experiment with the ternary complexes formed with MgADP and
ADP with ScF
and with MgADP and Vi (as a control)
are presented in Fig. 5. It is clear that actin binding
dissociates the ternary complex and results in the regain of the EDTA
ATPase activity. The actin-induced rates of decomposition of these
complexes can be roughly estimated from the half-life of regain of full
ATPase activity. This gives a lower limit for the observed rate of
complex decomposition (k
), and the values were
found to be 1.28
10
s
for
both the MgADP-containing complexes trapped by Vi or
ScF
. This is in reasonable agreement with the data
of Werber et al.(1992) for the MMgADP
Vi complex under
similar conditions. In contrast, the ADP-containing complex formed with
ScF
is decomposed more slowly by actin at a rate
of about 0.29
10
s
.
Figure 5:
Effect of actin on the inhibitory complex.
F-actin was added to a final concentration of 10 µM, and
the S1 complexes were 5 µM. The solutions were adjusted to
1.0 mM MgCl. The samples were incubated at 25
°C, and the release of ADP was estimated by the regain of the
NH
/(EDTA) ATPase at the time intervals
indicated.
, Vi;
, ScF
(MgCl
);
,
ScF
.
It
is well established that the tryptophyl fluorescence of S1 is sensitive
to the state of the bound nucleotide, and, therefore, the intrinsic
tryptophyl fluorescence of S1 in the presence of MgADP and NaF showed
an enhancement of about 7.0% over that of S1 alone. Addition of
Sc caused an additional enhancement of 10%
corresponding to a 17% enhancement of that for S1 in the absence of
nucleotide. This is close to the value of 20% observed for the addition
of MgATP to S1 and suggests that similar if not identical conformations
exist for the MMgADP
ScF
and the
MMgADP
P
complexes. In the absence of
MgCl
, addition of ADP to S1 in the presence of NaF resulted
in a 2% enhancement of the tryptophyl fluorescence, which upon addition
of the Sc
resulted in an additional enhancement of
14.6%, corresponding to a total enhancement of 16.6% over that of free
S1. Thus, both the magnesium-containing and magnesium-free complexes
stabilized by ScF
show very similar if not
identical levels of intrinsic tryptophyl fluorescence.
Cremo et
al.(1989) have shown that irradiation of the MMgADPVi
complex results in photo-oxidation of Ser-180 which is located in the
Walker A consensus sequence of some ATP-requiring proteins. This result
indicates that the bound Vi is located within chemical bond length of
this seryl residue. In the absence of MgADP, irradiation of S1 in the
presence of Vi results in cleavage at sites located at 23, 31, and 74
kDa from the N terminus with those at 23 and 31 kDa attributed to Vi
binding to the same consensus sequence in S1 and a proximal serine,
probably Ser-243 (Ringel et al., 1990). These cleavages give
rise to bands of 74, 51, 46, and 23 kDa in SDS gel electrophoretograms.
Addition of Vi to the ternary complexes formed with
ScF
, and subsequent irradiation by near-UV light
showed cleavage only at the 74-kDa site (Fig. 6). The failure to
observe bands of 31 and 23 kDa in the two forms of ScF
complexes upon irradiation in the presence of Vi suggests
that Vi is unable to bind to the consensus sequence site due to the
occupancy by the ScF
anion.
Figure 6:
SDS-polyacrylamide gel electrophoresis
analyses of the effect of Vi-induced photolysis of S1 and of the
inhibitory complexes formed with ScF and MgADP or
ADP. Lanes: a, S1 with Vi (1.0 mM); b, S1
MgADP
ScF
with Vi (0.5
mM); c, same as b except Vi was 1.0
mM; d, S1
DP
ScF
with
Vi (0.5 mM); e, same as d except Vi was 1.0
mM; f, control S1.
The present study was undertaken to examine whether fluoride
complexes of a transition metal cation such as scandium could also
mimic BeF and AlF
in trapping
MgADP in S1 to form a stable complex at low temperature. The reasons
for choosing scandium were as follows. (i) The stability of the
fluoroscandium complexes is similar to fluoroaluminates (Goldstein,
1964) which have already been used to trap MgADP in S1. (ii) The
exchange rates of ligands for scandium lie between those of beryllium
and aluminum ions (van Eldik, 1986; Wilkins, 1991). (iii) We were
interested to determine whether the hexacoordinated scandium anions,
which may be in a deformed octahedral configuration (Nowacki, 1939),
would be effective in the protein to trap MgADP or free ADP. The data
presented in Fig. 1and Fig. 2indicate that
fluoroscandium anion (presumably ScF
) can
indeed produce an inhibitory complex of S1 in the presence of MgADP.
Surprisingly, inhibition was also observed when Mg
ions were omitted from the incubation mixture, indicating that
ADP can also be trapped in S1 by fluoroscandium. Direct chemical
analyses of the total amount of magnesium in solutions of S1 (13
µM) in the absence of MgCl
yielded a maximum
of 3.2 µM magnesium content, while in solutions of the
purified complex (at 13 µM) formed in the absence of
magnesium, the concentration of total magnesium was less than 2.0
µM which would yield a total concentration of less than
0.5 µM in MgADP in these solutions which is far too low to
account for stoichiometric trapping of MgADP in the protein.
Analysis for the amount of scandium present in these complexes
indicate that 2 mol of scandium are present per mol of S1. The reason
for this is not as yet apparent. It is, however, possible to rule out a
high affinity site for the binding of ScF in the
absence of ADP, since little binding was observed when ADP and
MgCl
were absent (see ``Materials and Methods'').
It is unlikely that any of the bound scandium is in the form of
Sc
based on the association constants for the
formation of the fluoroscandium species in the presence of the high
concentrations of NaF employed (Goldstein, 1964). Therefore, it is
highly improbable that the complex formed in the absence of MgCl
involves any substitution of Sc
for
Mg
in coordinating to the bound ADP. We have also
found that inhibition occurs with BeF
and
AlF
with ADP in the absence of free
Mg
, but much higher concentrations of the fluorometal
anions are needed. (
)
The apparent second order rate
constants (k) for the onset of inhibition, 0.056 M
s
in the presence of
MgATP and 8.7 M
s
for
preincubation in its absence, are far too slow for a simple collisional
process of ScF
with S1
MgADP and suggest that
a very slow rate-limiting isomerization step occurs after formation of
the collisional complex as originally suggested by Goodno (1979) for
the Vi-induced inhibition of S1 in the presence of MgADP.
The
stability of the ternary complexes formed with fluoroscandium anion and
MgADP or ADP at 4 °C was followed by the release of the trapped C-nucleotide by chasing the isolated complexes with cold
ADP as described in Fig. 4. The time dependence of the release
indicated that it was biphasic for both forms of the complex. A similar
biphasic release was also observed with the complex stabilized with
BeF
(Phan and Reisler, 1992). For the two
ScF
complexes, the fast phase appeared to be
identical (k
= 8.0
10
s
) but the fractions of S1
associated with the fast phase were different being 0.46 and 0.11 for
the MgADP-containing and ADP-containing complexes, respectively. The
rates of the slow phases were also found to be different for these
complexes. The biphasic nature of the release of nucleotide from these
ternary complexes suggests the possibility that the ternary complex may
be in equilibrium between two distinct conformations, one in which the
nucleotide can be released more rapidly and a form where the nucleotide
is more strongly trapped. A similar mechanism has been proposed by
Mihashi et al.,(1990) for the MMgADP
Vi complex. In that
study it was shown, using an exchange procedure, that nucleotide could
undergo exchange with trapping, suggesting that release of nucleotide
from the inhibitory equilibrium mixture preceded release of the Vi
anion. Furthermore, the rate constant of the rapid phase for the
exchange in the case of the Vi-stabilized complexes was 4.5
10
s
which is about a factor of 6
times greater than that observed for the release of ADP from the
ScF
-trapped MgADP in S1 measured at 4 °C. The
rates of the slow phase of releases of ADP from the MgADP-containing
ScF
and ADP-containing complexes, 8.0
10
s
and 3.9
10
s
, respectively, are in
reasonable accord with the slow rate of exchange measured for the
MMgADP
Vi complex (6.7
10
s
) (Mihashi et al., 1990).
The
enhancement of tryptophyl fluorescence associated with the formation of
the two complexes (presence and absence of MgCl) is quite
similar, allowing for experimental error, and shows levels of
enhancement over that of unliganded S1 of about 17 ± 2%. This is
somewhat lower than that observed for the MMgADP
P
complex (20% ± 2%) but suggests that the conformations of
the complexes may be closely related to this steady-state intermediate.
The failure to detect any photocleavage at the two sites (21 and 31 kDa
from the N terminus) in the presence of added Vi for the two complexes
indicates that Vi probably does not bind to the region of S1 containing
the ATP consensus sequence (Walker et al., 1982), since it is
thought that occupancy of this region by Vi is essential for the
photocleavage to occur (Werber et al., 1992). This observation
is essentially identical with that noted for the complexes stabilized
with BeF
and AlF
,
respectively, and would be consistent with the ScF
occupying this site in the inhibitory complexes and the
inability of the added Vi to displace it (Werber et al.,
1992).
Perhaps the main question concerning these inhibitory
complexes stabilized by the various P analogs is to what
extent do they mimic the MMgADP
P
state. The large
enhancement of the tryptophyl fluorescence accompanying their formation
and the weak actin binding are in accord with their attaining a
structure similar to this steady-state intermediate. However, these
correlations may be coincidental. Another possibility is that, while
the initial collisional complex may be related to the
MMgADP
P
state, the much slower isomerization step
leading to inactivation may involve a rearrangement of the ligands in
the protein. For example, there is some evidence that ADP may interact
weakly with BeF
even in the presence of
Mg
(Issartel et al., 1991). While formation
of such a complex may be poorly favored in free solution, it is
conceivable that in the protein such a complex may be stabilized. While
it is usually assumed that the ligands bound in these inhibitory
complexes are present in a fashion analogous to those in the
MMgADP
P
complex, that is they are in the
M
MgADP
ScF
state, there is no direct
evidence to our knowledge that can rule out the possibility that the
slow isomerization leading to inhibition does not form a complex of the
form M
ScF
MgADP in which the nucleotide
is locked not at the active site but at another site due to a
rearrangement. In such a situation the complex would still be
inhibitory with the P
analog blocking access to the ATPase
site. Some support for this view is the recent finding that
photoaffinity-labeled S1, formed by irradiation of complexes trapped in
S1 by Vi, containing covalently linked nucleotide analog show normal
levels of ATPase, suggesting that the catalytic site was not labeled in
these complexes (Luo et al., 1994). (
)Secondly the
ability of the Vi-stabilized complexes to undergo nucleotide exchange
with trapping indicates that nucleotide release is faster than that of
Vi (Mihashi et al., 1990), and this is exactly the opposite
order of release occurring in the normal hydrolytic cycle with MgATP
(Bagshaw and Trentham, 1973, 1974).
In summary, the evidence
presented in this paper suggests that ScF is
capable of trapping either MgADP or ADP in S1 resulting in the
formation of rather stable inhibitory complexes (at 4 °C) and
extends the range of P
-like anionic analogs that are able
to function in this manner. Perhaps the most surprising outcome of the
present work is that free ADP is also apparently effectively trapped by
this anion in S1. The two complexes, with MgADP or ADP, show clear
differences in their stabilities at 4 °C and at 25 °C in the
presence of actin, and, in fact, the complex formed with ADP appears to
have the higher stability.