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INTRODUCTION |
The contraction of vertebrate striated muscle is controlled by the
thin filament-associated proteins, tropomyosin
(Tm)1 and troponin (Tn),
which regulate the interaction of actin and myosin in a
Ca2+-dependent fashion (1). In the absence of
Ca2+, Tm appears to bind to subdomain 1 of actin and bridge
over subdomain 2 to the next protomer, thereby blocking myosin access
to the strong binding sites on actin (2, 4-6). In the presence of Ca2+, Tm moves toward subdomain 3 and 4 (4, 6, 7), and this motion is completed upon binding of S1 (8, 9).
Based on structural models of regulated F-actin (2-5, 8, 10, 11),
subdomain 2 does not seem to have a marked role in interactions with
regulatory proteins. However, Squire and Morris (12) raised the
possibility that changes seen in the structural studies of regulated
thin filaments may be due not only to shifts of the Tm/Tn complex, but
also to subdomain 2 movements within each actin protomer. The
observations that selected subdomain 2 alterations impair regulation by
troponin-tropomyosin (13, 14) and that the presence of Tm and Tm/Tn
interferes with cross-linking between subdomain 2 and S1 (15) are
consistent with this hypothesis. Moreover, the recent paper of Luo
et al. (16), reporting the cross-linking of Tn-I to
Met47 on subdomain 2 of actin, suggests that TnI interacts
with this region of actin. A similar conclusion was reached in an
electron microscopic study of thin filaments containing mutant
tropomyosin, in which troponin density was found near Met47
(although troponin more strongly contacted subdomain 1 than subdomain 2) (7). Although previous work from our laboratory (17) did not detect
any subdomain 2 movements during regulation, all of our experiments
used probes attached to Gln41 (in
-skeletal actin) or
Cys41 and Cys51 (in mutant yeast actins).
Because such probes may have altered the dynamics, if not the
conformation, of an intrinsically dynamic subdomain 2, and because TnI
cross-links to Met47 on actin, we tested for Tm/Tn
interactions with this region using subtilisin-cleaved actin (3,
18).
Subtilisin cleaves between Met47 and Gly48 in
loop 38-52 of actin's subdomain 2 (3). The resultant protein exhibits
modified interprotomer interactions in F-actin and impaired function
with myosin (i.e. myosin binding, acto-S1 ATPase, and
in vitro motility) (3, 18). Since these changes occur
subsequent to subdomain 2 cleavage, the question addressed in this work
is whether, and to what extent, the subtilisin-cleaved actin is still
regulated by Tm/Tn. Alterations in regulation would indicate that
subdomain 2 indeed has a direct or indirect role in regulation.
Previous studies have shown that the presence of the Tm/Tn regulatory
complex not only introduces Ca2+-based regulation to
F-actin filaments but also potentiates their function, i.e.
the in vitro motility of actin filaments and the force
generated by actomyosin are enhanced (19-21). Depending on the assay
conditions and the types of Tm and Tn used, thin filament sliding speed
increases of up to 100% have been reported (19-22). Although several
explanations for such actin speed increases can be considered, the most
likely possibility is that of accelerated actomyosin detachment through
faster ADP release and/or ATP binding. Because the stopped flow
technique limits measurements of the kinetic rates of these processes
to 500-700 s
1, the effect of Tm/Tn on these rates is not
easily measured. Such measurements, however, are feasible using the
cleaved actin, and they were carried out in this study.
In the experiments described below, we investigated the effect of
regulatory proteins on the in vitro motility of
subtilisin-cleaved actin, the binding of S1 to such actin, and the rate
of ADP release from S1 bound to actin. We compared the degree of
protection of myosin loop 1, which is part of the myosin nucleotide
binding cleft, afforded by cleaved and uncleaved actin. Our findings
indicate a modulation of cross-bridge cycle kinetics by the Tm/Tn
regulatory complex, while also supporting the conclusion that subdomain
2 conformation does not play an essential role in Tm/Tn-based
regulation of actomyosin interactions.
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MATERIALS AND METHODS |
Reagents--
ATP, ADP, DTT, phalloidin, and PMSF were purchased
from Sigma. N-(1-pyrene)-maleimide was purchased from
Molecular Probes, Inc.
Proteins--
Skeletal myosin and actin were prepared from
rabbit back muscle according to Godfrey and Harrington (23) and Spudich
and Watt (24), respectively. S1 and heavy meromyosin (HMM) were prepared from myosin using the protocols of Weeds and Pope (25) and
Kron et al. (26), respectively. The cardiac troponin and tropomyosin were produced as previously described by Tobacman and
Adelstein (27). Subtilisin was purchased from Sigma.
The labeling of skeletal actin with pyrene maleimide was performed
according to a previously described protocol (28). The extent of
labeling was ~100%.
Actin was cleaved by adding subtilisin to skeletal G-actin in a 1:1000
weight ratio. The cleavage was carried out at room temperature for 15 min at 20 °C. The reaction was terminated by the addition of PMSF to
1.0 mM. The cleaved actin was then polymerized by raising
the MgCl2 and KCl concentrations to 2.0 mM and
50 mM, respectively. The cleaved G-actin was run on a
12.5% SDS-polyacrylamide gel to verify the complete cleavage and was
used in experiments within 24 h after the cleavage.
Acto-HMM ATPase Measurements--
The rates of HMM
Mg-ATPase were determined at 25 °C under steady-state
conditions, by monitoring the inorganic phosphate release after ATP
hydrolysis as described before (18). Thin filaments were reconstituted
using bovine cardiac troponin, rabbit skeletal tropomyosin, and the
subtilisin-cleaved or intact skeletal actin. The assay solutions
contained 10 mM imidazole-HCl, pH 7.0, 10 mM
NaCl, 2.0 mM K+-EGTA, 3.0 mM
Mg2+-ATP. Protein concentrations were: 0-8
µM for HMM (0-16 µM S1 heads) and 0-40
µM actin (intact and cleaved).
Competition Binding Experiments--
The binding of S1·ADP to
subtilisin-cleaved and pyrene-labeled actins was monitored by measuring
the quenching of pyrene fluorescence by S1 in samples containing 1.0 µM pyrene-labeled actin and comparing them with samples
containing 1.0 µM pyrene-labeled actin and 5.0 µM subtilisin-cleaved actin with and without regulatory
proteins (14). When present, the molar ratios of Tm and Tn to actin
were 0.35:1.0. S1 was added to actin solutions in 0.25-µM
increments to a total concentration of 3.0 µM. To ensure
that all binding was strong (in the absence of ATP), 5 units of
hexokinase and 1.0 mM glucose were added to each sample to
hydrolyze any contaminant ATP. The assay buffer also contained 2.0 mM ADP, phalloidin in equimolar concentrations with the
actins, 4.0 mM MgCl2, 150 mM NaCl,
and 2.0 mM DTT. All measurements were recorded at 23 °C in Spex Fluorolog using excitation and emission wavelengths of 365 nm
and 405 nm, respectively. Dissociation constants were determined using
the program Nfit 1.0 (University of Texas, Galveston, TX) and fitting
the data with the equation
|
(Eq. 1)
|
where S0 represents total S1 concentration,
Kd,P = dissociation constant for
pyrene-labeled actin; F = fluorescence;
F0 = maximum fluorescence, i.e. the
fluorescence of pyrene-labeled actin before the addition of S1;
A0,P = concentration of pyrene-labeled actin;
FS = minimum fluorescence, after saturation with
S1; A0,S = concentration of subtilisin-cleaved actin;
Kd,S = dissociation constant for
subtilisin-cleaved actin.
The above equation, connecting the measured fluorescence with
the concentrations of the bound and free actins, was derived by
algebraic manipulation of the following expressions,
|
(Eq. 2)
|
where S0 represents total S1 concentration, S is the
unbound S1, and APS and ASS represent S1
complexed with pyrene-labeled and subtilisin-cleaved actin,
respectively. Each of these species is expressed in terms of a
dissociation constant (Kd), using the mass
action law relationships
|
(Eq. 3)
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and
|
(Eq. 4)
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and is related to the observed fluorescence (F) via the
following equation.
|
(Eq. 5)
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In Vitro Motility Assays--
The in vitro motility
assays were performed according to a previously described protocol
(29). Tm and Tn, when present, were added to the assay system at the
concentration of 0.1 µM; Tm alone was present at 0.4 µM in these assays. Movement was initiated by applying an
assay buffer (2.0 mM
K+-EGTA/Ca2+-K+-EGTA (with the
ratio varying depending on the pCa desired), 20 mM KCl, 2.0 mM MgCl2, 10 mM DTT, 25 mM MOPS, pH 7.3; total ionic strength, 50 mM) containing 1.0 mM
Mg2+-ATP and an oxygen scavenging system (30) at
25 °C. An ExpertVision System (Motion Analysis, Santa
Rosa, CA) was used to quantify the sliding speeds of individual
filaments. Actin speeds (mean ± S.D.) were determined from
measurements of motion of 150-250 filaments. Individual filaments were
judged to be moving smoothly and were used for statistical analysis if
the standard deviation of their sliding speeds was less than one-half
of their average speed (31). Filament speeds at different
pCa values were fitted to a Hill equation in the form,
|
(Eq. 6)
|
where V is the measured mean speed of actin filaments,
V0 is the mean filament speed at pCa = 5.0 (maximal speed), pCa50 is the calcium
concentration at which V = 0.5 V0, and n is
the Hill coefficient. Significance was determined using Student's t-test, and the confidence level was set at
p < 0.05.
Stopped-flow Experiments--
The stopped-flow measurements were
carried out at 20 °C in a buffer containing 100 mM KCl,
25 mM MOPS, pH 7.3, 2.0 mM MgCl2, 2.0 mM Ca2+-K+-EGTA, 2.0 mM DTT, and 1.0 mM PMSF. The final
concentration of S1, F-actin (whether cleaved or uncleaved), and ADP
(when present) were 2.0, 3.0, and 100 µM, respectively.
Phalloidin was added at an equimolar concentration to actin for
filament stabilization. When applicable, rabbit skeletal Tm and bovine
cardiac Tn were each present at a concentration of 0.75 µM. The final concentration of Mg·ATP, after mixing,
was 5.0 mM. The rate-limiting dissociation of ADP from
acto-S1 was followed via a decrease in light scattered at 90 ° to
the exciting light that accompanied the subsequent ATP-induced
dissociation of the acto-S1 complex. Excitation and emission
wavelengths were both 345 nm.
Papain-digestion Assays--
Papain was activated by dissolving
it in a solution containing 50 µM DTT, 1.5 mM
EDTA, and 5.5 mM cysteine, pH 6.0, and incubating for 30 min on ice before use. The digestions were performed at room
temperature, at S1 concentration of 8.7 µM and, where
applicable, F-actin concentration of 17.4 µM. Papain was
added to each sample to a final concentration of 50 µg/ml, and the
digestions were terminated by adding 10 mM iodoacetic acid.
The extent of S1 digestions lasting 0, 5, 10, 15, 25, and 40 min was
estimated from scans of SDS-PAGE gels.
 |
RESULTS |
Actin Sliding in the in Vitro Motility Assays--
As documented
earlier (3, 18), our limited digestion of actin by subtilisin yielded
homogenous preparations with virtually no uncleaved or farther degraded
actin. As also reported before (18), the in vitro motility
of subtilisin-cleaved actin was strikingly impaired. The results
of these measurements are listed in Table
I. The in vitro motility
speeds for the cleaved and uncleaved actins without regulatory proteins
were 1.9 ± 1.1 and 4.1 ± 0.7 µm/sec, respectively. We
found that in the presence of Ca2+, at pCa = 5.0, the regulatory complex increased the sliding speed of the
subtilisin-cleaved and uncleaved actin filaments by about 2-fold, to
3.57 ± 1.2 and 7.45 ± 1.1 µm/s, respectively (Table I).
The presence of the regulatory proteins also increased the percentage
of smoothly moving cleaved actin filaments ~2.5-fold, from 34.4% to
88.5%. Tm alone had a much smaller effect than Tm/Tn on the motility
of the cleaved and intact actin (Table I). In the presence of Tm the
speeds of actin increased by up to 20% (± 10%).
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Table 1
In vitro motility speeds of intact and subtilisin-cleaved actin in
presence and absence of regulatory proteins
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Measurements of the sliding speeds of regulated actin versus
pCa revealed similar dependence on Ca2+ for the
cleaved and intact actin (Fig. 1,
inset). This is easily seen after normalizing the actin
filament speeds to 100% at pCa = 5.0 (Fig. 1). Thus,
the regulatory mechanism seems little affected by the cleavage; both
the cleaved and uncleaved actins have similar pCa50 and Hill coefficients (Fig. 1).

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Fig. 1.
Effect of pCa on the
in vitro motility of regulated uncleaved and
subtilisin-cleaved actin filaments. The speeds and numbers of
smoothly moving actin-Tm/Tn filaments were measured at 25 °C
as they moved over a surface coated with HMM. The motility assay buffer
contained 0.1 µM Tm, 0.1 µM Tn, 2.0 mM K+-EGTA/Ca2+-K+-EGTA
(with the ratio varying depending on the pCa desired), 20 mM KCl, 2.0 mM MgCl2, 10 mM DTT, 25 mM MOPS, pH 7.3, 1.0 mM
Na2ATP (total ionic strength, 50 mM) 14 mM glucose, 9 × 103 units catalase/ml,
and 240 units of glucose oxidase/ml (26). The normalized speeds of
uncleaved ( ) and subtilisin-cleaved ( ) actins are plotted
versus pCa, with the data normalized based on the
speeds at pCa 5.0. Inset, actual speeds of
uncleaved and subtilisin-cleaved actins. Error bars
represent standard deviation from the mean speeds at each
pCa value. The data were fitted to the Hill equation (see
"Materials and Methods") with R2> 0.95. The
uncleaved actin ( ) (upper trace) moved at
pCa = 5.0 at a mean speed of 7.45 ± 0.56 µm/sec, a Hill coefficient of 2.67 ± 0.87, and a
pCa50 of 6.63 ± 0.06 under the same
conditions. Cleaved actin ( ) (lower trace) moved at
pCa = 5.0 at a mean speed of 3.57 ± 0.36 µm/sec, a Hill coefficient of 2.18 ± 0.81, and a
pCa50 of 6.61 ± 0.09.
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S1 Binding--
The affinity of S1 to pyrene-labeled skeletal
actin and subtilisin-cleaved actin in both the presence and absence of
Tm/Tn was measured in binding competition assays. The goal of these measurements was to determine the effect of the regulatory proteins on
the affinity of cleaved actin for S1 and to assess the possibility that
Tm/Tn might "restore" their binding to that of intact actin and S1.
The presence of the regulatory complex increases the affinity of the S1
for the pyrene-labeled actin by about 5-fold, with the Kd decreasing from 0.18 ± 0.003 to 0.037 ± 0.003 µM (mean ± S.E.) in the absence and
presence of Tm/Tn, respectively (Fig. 2).
A similar effect of Tm/Tn on the affinity of S1 to pyrene-labeled actin
was reported by Korman et al. (14). The cleaved and
unlabeled actin bound S1 less well than the intact labeled actin, with
a Kd of 0.39 ± 0.006 µM. Taking
into account the ~5-fold increase in the Kd value
of S1 and actin due to pyrene labeling of actin (14), our measurements
show about a 10-fold lower binding constant of S1 to cleaved than to
intact actin. This result is consistent with prior measurements of the
effect of subtilisin cleavage of actin on S1 binding (3). In the
presence of regulatory proteins, the affinity of S1 to cleaved actin
increased by 2-fold, with a Kd of 0.22 ± 0.004 µM. This improvement in binding is less than the 5-fold
effect of troponin-tropomyosin measured using the uncleaved labeled
actin. Thus, even in the presence of Tm/Tn the cleaved actin binds S1
less strongly than intact actin in the absence of regulatory
proteins.

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Fig. 2.
S1·ADP binding to uncleaved and cleaved
actins with and without regulatory proteins. The competition
experiments were done by measuring the quenching of pyrene-labeled
actin fluorescence by S1. Two samples containing 1.0 µM
pyrene-labeled actin, with ( ) and without ( ) Tm/Tn, were compared
with two samples containing 1.0 µM pyrene-labeled actin
and 5 µM subtilisin-cleaved actin, also with ( ) and
without ( ) regulatory proteins (when present, the molar ratios of Tm
and Tn to actin were 0.35:1.0). The assay buffer was made up of 2.0 mM ADP, phalloidin in equimolar concentrations with the
actins, 4.0 mM MgCl2, 150 mM NaCl,
and 2 mM DTT with 5 units of hexokinase and 1 mM glucose added to each sample to hydrolyze any ATP that
may have been present. All measurements were recorded at 23 °C using
excitation and emission wavelengths of 365 and 405 nm
respectively.
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Besides decreasing the affinity of S1 binding, subtilisin cleavage of
actin also decreases weak acto-S1 interactions as evidenced by its
increase of the Km (from 33 to 200 µM)
value of the actomyosin ATPase reaction (3, 18). We verified in
acto-HMM ATPase measurements that the difference between the
Km values of cleaved and intact actin in such assays
is retained in the presence of Tm/Tn (at pCa = 5.0;
data not shown). Taken together, the small effect of Tm alone on the
motility of cleaved actin, and the S1 binding and acto-HMM ATPase
results indicate that the regulatory proteins do not offset the
cleavage damage to the interaction with myosin.
Stopped-flow Measurements of ADP Release from Regulated and
Unregulated Acto(cleaved)-S1--
Stopped-flow measurements of the
ATP-dependent rate of dissociation of rigor acto-S1 complex
at 20 °C did not reveal any major slowing of this process when using
cleaved actin. The rates of dissociation of S1 from both cleaved and
uncleaved actin were linear with the [Mg·ATP] and were >800
s
1 at 5 mM ATP (the highest rate we could
reliably measure). Such fast rates preclude the measurement of any
possible effect of regulatory proteins on acto-S1 dissociation.
Although lowering the temperature of the solution would slow acto-S1
dissociation, this strategy is not useful because regulatory proteins
loose their ability to potentiate thin filament sliding speeds at low temperatures
(<12 °C).2
Stopped-flow measurements of ADP release from S1 at 20 °C in the
presence of both regulated and unregulated subtilisin-cleaved actin
filaments were fitted using a single exponential equation. The results
reveal an almost 8-fold increase in the rate of ADP release by the
regulated compared with the unregulated cleaved actin, with release
rates of >900 s
1 and 125 ± 12 s
1,
respectively (Fig. 3). We were unable to
reliably measure the release rates of ADP from uncleaved acto-S1 as
these rates were beyond instrument resolution, even without regulatory
proteins (>850 s
1). The nucleotide release rates can be
decreased considerably by using the ADP analog
ADP in
lieu of ADP in such experiments. The results of such
measurements showed that the rates of
ADP release from S1 are about
the same in the presence of unregulated and regulated actin (32, 33).
Our in vitro motility experiments using
ATP instead of
ATP also did not reveal any effect of Tm/Tn on actin speeds. In the
presence of
ATP unregulated and regulated intact actin moved at
1.37 ± 0.45 and 1.29 ± 0.41 µm/s speeds (mean ± S.D.), respectively.

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Fig. 3.
ATP dissociation of cleaved
acto-S1·ADP. A shows the time course of the decrease
in light scattering from cleaved acto-S1·ADP mixed with 5.0 mM Mg·ATP. The light scattering trace was fitted by a
single exponential whose rate was 125 ± 12 s 1.
B shows the dissociation of cleaved acto-S1·ADP in the
presence of bovine cardiac troponin, rabbit skeletal tropomyosin, and
5.0 mM Mg·ATP. The light scattering trace was fitted by a
single exponential with a rate 965 ± 35 s 1. In each
case R2 > 0.95. Each reaction time
course is the average of three to five separate recordings. The
solid smooth lines are exponential fits to the data with the
indicated rates.
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Protection of Loop 1 in S1 from Proteolysis: Cleaved versus
Uncleaved Actin--
Modification of loop 1 of S1 has been shown to
affect the rate of nucleotide release from acto-S1 (34-36). In
addition, despite the fact that actin does not directly contact this
area of S1, it protects S1 loop 1 from digestion by papain (37). By
comparing the degree of protection afforded by cleaved
versus uncleaved actin, we sought to determine whether the
actin cleavage by subtilisin affected the ability of actin to modulate
this site. However, the degree of protection from papain proteolysis
afforded S1 by the cleaved actin was virtually the same as that by
uncleaved actin (Fig. 4). This shows that
actin cleavage alters ADP release from acto-S1 by weakening the impact
of actin on structural elements of S1 other than loop 1.

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Fig. 4.
Limited proteolysis of S1 by papain as
monitored by SDS-PAGE. Lanes 1-4 show controls of S1 alone
digested with papain for 0, 5, 15, and 40 min, respectively. Actin/S1
(lanes 5-8) and cleaved-actin/S1 (lanes 9-12)
are shown for the same digestion times as the control. As lanes
5-8 and 9-12 indicate, both actins protect S1 loop 1 from papain digestion equally well. Protein bands are identified on the
right side of the gel panel. cS1 corresponds to the
papain-cleaved S1 band.
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|
 |
DISCUSSION |
Recently, Luo et al. (16) reported on the
photocross-linking of residues 104 and 133 on mutant troponin I (TnI)
to Met47 on actin in reconstituted thin filaments in the
absence of Ca2+. On this basis, Luo et al. (16)
suggested that TnI binds to actin in the vicinity of Met47,
probably to the adjacent segment 50-58. Binding of this actin region
to TnI may form a Tm interaction site, which can stabilize the latter
protein at the outer domain of actin i.e. in the blocked state. In the presence of Ca2+, the release of TnI from its
binding region on actin would allow the movement of Tm to its
thermodynamically preferred position between the outer and inner
domains of actin, i.e. to the closed state position (16).
This regulation model does not evoke any companion dynamic changes on
actin and, therefore, is not in conflict with the apparent absence of
subdomain 2 motions during actin regulation (17).
The proposed location of the TnI binding site on actin (segment 50-58)
and the easy availability of actin specifically cleaved by subtilisin
between Met47 and Gly48 (3, 18) suggested that
such material could be used to examine the dependence of regulation on
subdomain 2 interprotomer contacts, the conformational changes in this
region, and the role of subdomain 2 in transmission of regulatory
allosteric changes to other parts of actin.
Several aspects of actin structure and function are impaired by its
subtilisin cleavage. These include the previously described (3, 18) and
confirmed above degradation of interactions with myosin, increased
critical concentration for polymerization (3), and decreased
inter-strand interactions (38). The cleaved actin filaments are also
more susceptible to tryptic proteolysis (at Lys61 and
Lys68), indicating a more open conformation of actin (39)
and/or greater mobility of its subdomain 2 within the polymer. It is possible that the decreased interactions of cleaved actin with myosin
are due to either a change in the binding site for myosin (if it
includes residues 38-52) or to a severed communication pathway in
actin by the cleavage between Met47 and
Gly48.
Despite changed interprotomer interactions in F-actin and the
functional impact of DNase-I binding loop cleavage, the regulatory mechanism was not impaired in the cleaved actin. When compared with
normal thin filaments, the degree of Tm/Tn-induced potentiation remained the same, with no significant shift in pCa
dependence of the regulation of cleaved actin (Fig. 1). This indicates
that the propagation of allosteric regulatory changes in actin occurs despite impaired interprotomer interactions and that these effects may
be propagated through the regulatory proteins themselves. Thus,
subdomain 2 does not seem to play an active role in the regulation of
actomyosin interactions. If TnI binds to segment 50-58 on actin (16),
it may act as a mechanical latch for Tm rather than allosteric
modulator of subdomain 2 and its dynamics. This conclusion is in line
with the results of our previous study in which we used probes attached
to actin's subdomain 2 to monitor its conformational transitions
during the regulatory process.
The steric-blocking model of actomyosin regulation does not provide a
simple explanation for the Tm/Tn-induced potentiation of actin speed
and force production in the in vitro motility assay (19).
Variations in filament speed and force generation are generally
attributed to changes in the kinetics of ADP release and recruitment of
myosin heads by the thin filament. Since previous studies (19-22) have
indicated increases in both force and speed in the presence of Tm/Tn,
it appears that the presence of regulatory proteins affects both myosin
head recruitment and nucleotide release through direct interactions
between the regulatory proteins and myosin, interactions mediated by
actin, or both. An increase in myosin recruitment, which would increase
force generation because of a greater number of heads attached at a
given time, is suggested by the increased binding of S1 to actin in the
presence of Tm/Tn (27, 40-42). However, under conditions of HMM
saturation in the in vitro motility assay, increases in
unloaded filament sliding speed can not be produced by increases in
myosin head recruitment (43). Instead, filament sliding speed increases
are more likely produced by increased cross-bridge cycle turnover rate
or faster ADP release. This scenario is supported by a recent study of
Homsher et al. (19), showing that the enhancement of actin
speed by Tm/Tn is progressively lost by increasing the
[Mg·ADP].
We showed in our binding competition assays that the presence of
regulatory proteins increases the affinity of S1 to the thin filament
reconstituted with intact actin ~5-fold (Fig. 2). For thin filaments
containing cleaved actin, the increase in affinity is less than 2-fold.
These results indicate that the difference in the binding of myosin
heads by the two types of actin is not decreased in the reconstituted
thin filaments. Despite this difference, both types of regulated thin
filaments display 2-fold increases in the in vitro motility
speeds. This confirms the contention that increases in speed are not
tied to increases in recruitment, but are most likely due to increases
in the rate of ADP release. Consistent with this, when such
acceleration in ADP release does not occur, as in the case of
ADP
(32, 33), we have measured similar actin speeds with and without
Tm/Tn.
Due to the extremely rapid ADP release rate from acto-S1 at room
temperature, we were unable to determine the effect of Tm/Tn on the ADP
off rates from uncleaved actin/S1. Using lower temperatures to slow the
kinetics of this reaction was not an option as below 12 °C Tm/Tn
cause a decrease in the in vitro motility performance instead of the potentiation that is seen at room
temperature.2 However, given the performance of the
regulated subtilisin-cleaved actin filaments in the in vitro
motility assay, this system provided a convenient model for the study
of ADP release from the acto-S1 complex. As reported above, S1 in the
presence of unregulated cleaved actin filaments released ADP at a much
slower rate than in the presence of their uncleaved counterparts, but
this rate was increased severalfold upon the addition of Tm/Tn. Thus,
our finding provides an experimental link between the in
vitro motility speed increases in the presence of the regulatory
complex and the increase in the rate of ADP release from myosin.
The improved binding of S1 to cleaved actin in the presence of Tm/Tn,
which increases the S1 affinity to actin by less than 2-fold, cannot
account fully for the acceleration of ADP release. It appears that
while the cleaved actin fails to open the nucleotide cleft on S1 to the
same extent as the intact actin does, Tm/Tn restores that ability to
the cleaved protein. At present, it is unclear whether this effect is
due to direct Tm-S1 interactions or allosteric changes on actin
(induced by Tm/Tn) that potentiate its effect on the nucleotide cleft
on S1. To test one possible structure-function relationship behind the
effect of actin on ADP release from S1, we compared the ability of
intact and cleaved actins to protect loop 1, which spans the 25/50-kDa
junction of S1, from papain cleavage. Despite the fact that actin does
not directly contact this site, the binding of actin to S1 causes a
conformational change in loop 1, which inhibits its cleavage by papain.
Loop 1 is located over the nucleotide binding cleft, and various
studies have shown that modifying it affects nucleotide binding. Rovner
et al. (36) have shown a marked increase in the in
vitro motility of actin and suggested a concomitant increase in
ADP release when this loop is altered through mutations. In line with
this, Sweeney and Holzbaur (34) also demonstrated the postulated rate
increases in ADP release. Additional studies by the same laboratory
showed that the rate of ADP release is correlated to the length and
flexibility of loop 1 (35). Thus, given the reported effect of loop 1 on ADP release and our own results, which showed changes in motility
consistent with shifts in ADP release, we investigated the possibility
that subtilisin-cleaved actin had a different effect on loop 1 than the
intact actin. Our results indicate no changes in the degree of
protection provided by the cleaved actin. Thus, the mechanism involved
in the shift in ADP release brought about by the cleaved actin does not
involve changes in actin impact on loop 1 of S1.
In summary, our work shows that the presence of regulatory proteins
increases the speed of cleaved actin filaments in the in
vitro motility assay, and this effect is correlated with the increase in the rate of ADP release from myosin. Despite the functional differences between the modified and unmodified actins, actin regulation and the effect of the regulatory complex on speed is unchanged; it is unaffected by the impairment of interprotomer actin
interactions. While the cleavage in actin subdomain 2 does affect
interactions between actin and S1, these changes do not appear to
include the influence of actin on loop 1 in S1. Therefore, the
mechanism that alters the rate of ADP release must be due to other
changes in acto-S1 interactions.