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J Biol Chem, Vol. 274, Issue 25, 17545-17550, June 18, 1999
From the According to the Lorenz et al.
(Lorenz, M., Poole, K. J., Popp, D., Rosenbaum, G., and Holmes,
K. C. (1995) J. Mol. Biol. 246, 108-119) atomic model
of the actin-tropomyosin complex, actin residue Asp-311 (Glu-311 in
yeast) is predicted to have a high binding energy contribution to
actin-tropomyosin binding. Using the yeast actin mutant E311A/R312A in
the in vitro motility assays, we have investigated the role
of these residues in such interactions. Wild type (wt) yeast actin,
like skeletal The contraction of vertebrate striated muscle is regulated by the
thin filament-associated proteins tropomyosin
(Tm)1 and troponin (Tn),
which modulate the interaction of actin and myosin in a
Ca2+-dependent fashion (2). Each Tm molecule
associates with one Tn molecule and seven actin monomers. The amino
acid sequence of Tm contains a pattern of charged and uncharged amino
acids that repeats 14 times along its length (3). As each pair of repeats corresponds to an actin monomer along the actin filament, it
has been inferred that the binding of Tm to actin is dominated by
electrostatic interactions (1).
Tn is composed of three subunits: TnT, TnI, and TnC. The TnC subunit
binds Ca2+ and confers calcium sensitivity to the
actin-Tm-Tn system. According to the three-state model of thin filament
regulation (4), the actin-Tm-Tn complex can assume three structural
states: blocked, closed, and open. In the blocked state there is a very
low incidence of myosin binding. When Ca2+ binds to TnC,
the Tm-Tn complex shifts to the closed state, uncovering additional
myosin weak binding sites on the actin filament. The increase in weak
binding and the initial strong binding of myosin induce Tm-Tn to shift
from the closed (where it prevents myosin strong binding) to the open
state. According to this model, the azimuthal shift of Tm-Tn around the
axis of the actin filament sterically regulates myosin binding to actin.
Despite the elegance of the three-state model, it cannot explain well
the findings of earlier acto-S1 ATPase solution studies (5-7). Results
from these studies describe the ability of Tm alone to both inhibit, at
low S1 concentrations, actin-activated S1 ATPase rates and to
potentiate the reaction at intermediate, nonsaturating concentrations
of S1. Although inhibition can readily be explained using the steric
block model, the potentiation suggests the presence of an allosteric
component in actomyosin regulation.
The Ca2+-induced Tm-Tn movement on actin was first
indicated by x-ray diffraction studies (8-10). Electron microscopy has
also been used to directly visualize this shift in Tm-Tn position
(11-16), and the studies of Limulus muscle (17, 18) and
vertebrate muscle (19) identified the positions for the Tm-Tn complex
on actin in the presence and absence of Ca2+. A high
resolution model of the Tm-F-actin complex was proposed by Lorenz
et al. (1) on the basis of their x-ray fiber diffraction investigation. According to these authors, Tm alone and Tm-Tn in the
presence of Ca2+ reside in the same closed-state
orientation on the actin filament. In this study Lorenz et
al. (1) predict that 16 actin residues participate in the
electrostatic interactions between F-actin and Tm and calculate their
In this study, we compared the regulation and function of wt and
E311A/R312A yeast actins, as well as their interaction with Tm, in the
in vitro motility assays, equilibrium binding experiments, and acto-S1 ATPase measurements. The results of our work support an
allosteric explanation of the role of Tm in the regulation of
actomyosin interaction.
Reagents--
ATP, ADP, dextrose, DTT, phalloidin,
phenylmethylsulfonyl fluoride, and Proteins--
Skeletal myosin and actin were prepared from
rabbit back muscle according to Godfrey and Harrington (21) and Spudich
and Watt (22), respectively. S1 and HMM were prepared from myosin using
the protocol of Weeds and Pope (23) and Kron et al. (24), respectively. Yeast actins were purified over a DNase I affinity column
(25) and were stored on ice in a G-actin buffer (5 mM TES,
0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM Actin-activated ATPase--
The malachite green assay (29) was
used to measure the ATPase activity of actin-activated S1. The
procedure was the same as that used by Miller et al. (30).
The assays were carried out at 22 °C in a buffer containing 5 mM KCl, 2 mM MgCl2, and 10 mM imidazole, pH 7.4. The S1 concentration was 0.4 µM, whereas that of the mutant or wt actin ranged between
0 and 35 µM.
Regulated Actin-activated ATPase--
Hydrolysis rates of
regulated actin-activated S1 MgATPase at pCa values ranging between 5.0 and 9.0 were obtained by using light scattering to monitor the clearing
time of regulated F-acto-S1 solutions. Clearing time is defined as the
duration of the decrease in light scattering of acto-S1 solutions after
the addition of ATP. The light scattering of the solution increases
sharply upon the hydrolysis of the added ATP, leading to the
determination of clearing time, i.e. the time of ATP
hydrolysis. Thin filaments were reconstituted using either wt or
E311A/R312A actin, bovine cardiac troponin, and either bovine cardiac
or skeletal tropomyosin. The assay buffer was adjusted to 30 mM total ionic strength (including calcium concentrations)
using a program written in QuickBasic by Drs. E. Homsher and N. Millar
based on the equation of Fabiato and Fabiato (31). This buffer
contained 5 mM imidazole (pH 7.5), 13.2 mM KCl,
3 mM MgCl2 (free), 2 mM EGTA (with
varying ratios of
Ca2+-K+-EGTA/K2EGTA), and 15 mM DTT. The S1 concentration was 1.0 µM, whereas that of actin was 7.0 µM. The concentrations of
Tm and Tn were 2.0 and 1.2 µM, respectively. Experiments
were carried out at 23 °C using a MgATP concentration of 0.1 mM. The course of MgATP hydrolysis was monitored by
measuring the light scattering at 350 nm of the above solutions in a
Spex Fluorolog (Spex Industries Inc., Edison, NJ).
Actin Polymerization--
Polymerization of both the mutant and
the wt G-actins (5.0 µM) by MgCl2 (3.0 mM) was monitored by measuring the light scattering in a
Spex Fluorolog set at 325 nm.
Circular Dichroism and Tryptophan Fluorescence--
The CD
spectra of the monomeric actins were recorded between 190 and 250 nm in
a G-actin buffer (see above) at 25 °C using a Jasco J-600 CD
spectropolarimeter. Tryptophan fluorescence emissions spectra of the
G-actins were recorded at 23 °C in Spex Fluorolog using 295 nm as
the excitation wavelength.
Cosedimentation Assays--
Using cosedimentation assays, we
compared the strong binding of S1 (1-5 µM) to each of
the F-actins (4.0 µM) stabilized by equimolar
concentrations of phalloidin. The assays were performed at 22 °C in
a buffer made up of 3.0 mM MgCl2, 100 mM NaCl, and 10 mM imidazole at pH 7.4. The
samples were pelleted using a Beckman TL-100 centrifuge and TLA-100
rotor spinning at 75,000 rpm (217,000 × g) for 30 min.
Prespun, supernatant, and resuspended pellet solutions were run on
SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue.
The densities of the various bands were determined using an Arcus II
scanner (Agfa) and NIH Image version 1.60. The molar ratios of S1 bound
to actin and the binding constants (Ka) were
calculated by least squares regression using the program SigmaPlot 2.0 (Jandel Scientific). The binding of S1 to F-actin (3.0 µM) in the presence of Tm (3.0 µM) and
MgADP (3.0 mM) was measured as above, except for the lower
salt concentration used in the assay buffer (10 mM NaCl)
because of the reduced S1 binding to actin in the presence of MgADP.
In addition, we compared the binding of Tm to the actins in the
presence and absence of rigor-bound myosin S1. The assays were
performed using 5.0 µM actin (with equimolar
concentrations of phalloidin) with and without 10 µM S1,
0-2.2 µM N-(1-pyrenyl)iodoacetamide-labeled Tm in a 5.0 mM HEPES buffer, pH 7.5, 3.0 mM
MgCl2, 150 mM NaCl at 22 °C. The samples
were pelleted as above. The concentrations of
N-(1-pyrenyl)iodoacetamide-labeled Tm left in the
supernatants were determined by fluorescence measurements using a Spex
Fluorolog with excitation and emission wavelengths set at 344 and
385 nm, respectively.
In Vitro Motility Assays--
The in vitro motility
assays were performed according to a previously described protocol
(30). The HMM titrations are a variation of the above protocol in that
the HMM was applied to the nitrocellulose-treated coverslips at
concentrations ranging between 0.06 and 0.3 mg/ml. Regulated thin
filaments were assembled by incubating the rhodamine phalloidin-labeled
actin filaments (2.0 µM) with 0.5 µM each
of the regulatory proteins (Tm and Tn). The thin filaments were added
to the motility assay coverslip at 10 nM. After a 1-min
incubation, the unbound filaments were washed away with 50 µl of an
assay buffer containing 25 mM KCl, 1 mM EGTA, 4 mM MgCl2, 10 mM DTT, 0.1 µM of both Tm and Tn (or Tm alone, depending on the
nature of the assay), and 10 mM imidazole at pH 7.4. Regulatory proteins were included in the assay buffer to prevent the
dissociation of these proteins from regulated actin (32). Movement was
initiated by applying the same assay buffer containing 1.0 mM ATP and an oxygen-scavenging system (33). An
ExpertVision System (Motion Analysis, Santa Rosa, CA) was used to
quantify the sliding speeds of individual 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-third of their average velocity (34).
Ca2+ Titration of the Regulated System in the in Vitro
Motility Assays--
According to Lorenz et al. (1),
residue 311 has a considerable electrostatic role in the interaction
between actin and Tm in the closed state (
To test this possibility we measured the regulated thin filament
sliding speed at various pCa values for both the regulated mutant and
wt actins. Our results show a definite shift to the left of the pCa
curve for the 311/312 mutant, indicating a lowered dependence on
calcium to turn on the system (Fig.
2A). We also found a similar
trend in the numbers of moving filaments. At pCa 5.0 both actins have
approximately the same fraction of motile actin filaments. At lower
calcium concentrations, the regulated mutant actin has a larger
percentage of moving filaments than does wt and ceases to move at a
calcium concentration approximately half a pCa unit higher than that of
wt actin (Fig. 2B). Experiments similar to those shown in
Fig. 2 were repeated on four separate preparations of actins. In each
case the pCa shift of the mutant versus wt complex was
clearly defined. Although the midpoints of pCa titrations varied
somewhat among the preparations, the shifts between mutant and wt
actins were consistently reproduced (0.5 ± 0.1 pCa units).
Importantly, we saw no significant difference in sliding speed or
percentage of filaments moving between the two actins in the absence of
Tm and Tn (Table I).
As an additional control, we also performed motility experiments using
the yeast actin mutant K315A/E316A. According to the calculations of
Lorenz et al. (1), the electrostatic contribution of residue
315 is about 60% smaller than that of residue 311 ( In Vitro Motility of Actin-Tropomyosin Complexes--
Solution
studies have shown that the presence of Tm strongly influences the
acto-S1 ATPase rate; at low S1 concentrations Tm inhibits the ATPase
reaction, whereas at high S1 concentrations the reaction is potentiated
(5-7). According to biochemical and structural studies (1, 4), the
actin-Tm complex populates only two states, closed and open, of the
three states proposed for the Tm-Tn-actin complex in the McKillop and
Geeves (4) model. Clearly, the equilibrium between the closed and open
states of actin-Tm is shifted by the binding of S1. When S1 is present at sufficiently high concentrations, actin-Tm is switched to the open
state, releasing the inhibition of acto-S1 ATPase. To shed light on
these transitions and on the change in Ca2+ regulation of
the regulated mutant actin, we measured the in vitro
motilities of actin-Tm at various densities of HMM on the coverslips
used in these assays.
In the case of wt yeast actin we observed a Tm-induced slowing of actin
movement at low HMM concentrations (Fig.
3) but did not detect an acceleration of
actin sliding by Tm at high HMM concentrations. Tm did not increase the
sliding speed of wt thin filaments even when the switching "on" of
the actin filaments was facilitated by increasing the HMM concentration
to 0.5 mg/ml or by adding N-ethylmaleimide modified myosin
S1 to the motility assay buffer. However, our results for mutant
actin-Tm thin filaments (Fig. 3B) differ from those for wt
actin (Fig. 3A). Binding of Tm to the mutant thin filaments
significantly increased their sliding speed over surfaces incubated
with HMM at concentrations greater than 0.12 mg/ml. At the highest HMM
concentration tested, the mutant actin-Tm complex moved almost 50%
faster than the mutant actin alone (5.7 versus 3.8 µm/s).
On the other hand, at low applied HMM concentrations (0.06 mg/ml), the
mutant actin filaments behaved like their wt counterparts; the 311/312
actin alone was still sliding at speeds of approximately 1.8 µm/s,
whereas the actin-Tm complex did not move at all.
As expected, the percentage of filaments moving was high for both types
of actin at high applied HMM concentrations (data not shown). On
surfaces incubated with low HMM concentrations, however, the addition
of Tm significantly reduced the numbers of moving filaments. This
effect was especially marked for wt actin, with a significant drop in
the numbers of moving filaments at 0.10 mg/ml HMM and no moving
filaments noted at the lowest HMM concentration. The impact of Tm on
the mutant actin was less pronounced, with a clear decrease in the
number of moving filaments noted only at the lowest HMM concentration.
Thus, it appears that lower concentrations of HMM are required to
release the Tm-induced inhibition of actin sliding in the mutant than
in wt actin filaments.
Binding of Tm to Actin and S1·ADP to Actin-Tm--
In an effort
to relate the observed effects of Tm on actin motility to the binding
of Tm to actin, we measured the affinity of Tm for both the mutant and
wt actins using cosedimentation assays. The binding of Tm to both
actins was cooperative and could be described by sigmoidal curves with
Hill coefficients of 2.4 ± 0.4 for wt and 2.1 ± 0.6 for
311/312 actin (Fig. 4). There was no
significant difference in the binding coefficients, with
Ka values of (3.2 ± 0.2) × 106 M Structure-Function Studies--
Our results show significant
differences in the motility of complexes of Tm-Tn (fully regulated) and
Tm-alone with wt and 311/312 mutant yeast actin. Three possible
explanations for these differences come to mind: (i) the mutations
brought about changes in the actin structure which altered the function
of actin, (ii) the affinity of Tn for Ca2+ was impacted by
these mutations in actin, or (iii) the structure was not disturbed, but
the amino acid substitutions changed the interaction between actin and
the regulatory proteins. We performed a series of experiments to test,
and ultimately exclude, the first two possibilities.
Spectroscopic Assays--
Structural properties of the wt and
mutant actins were compared using CD and tryptophan fluorescence
measurements. Far UV CD spectra of 311/312 mutant and wt actins are
indistinguishable (Table I), indicating that the mutations have no
discernible effect on the secondary structure of actin. Similarly,
because the tryptophan emission spectra of the two actins are the same (Table I), the mutations do not appear to alter the tryptophan environments of the mutant actin.
Solution Interactions--
The rate at which actin polymerizes
from monomeric G-actin to polymeric F-actin is sensitive to its
substructure and conformational state (35-37). We compared the rates
of MgCl2-induced polymerization of both the 311/312 mutant
and wt actins by measuring the increase in light scattering of 5.0 µM G-actin solutions after the addition of 3.0 mM MgCl2. We found no significant differences
between the two actins (Table I).
The interactions of the mutant and wt actins with myosin were compared
in acto-S1 MgATPase measurements and in binding assays of the actins to
S1 under rigor conditions. The acto-S1 Mg-ATPase activities for wt and
311/312 mutant actins were virtually identical, yielding almost the
same Km and Vmax values
(Table I). Thus, at the very least, the 311/312 mutations affect
neither the weak binding interactions between actin and S1 nor the
activation of S1 MgATPase by actin. Moreover, the in vitro
motilities of the mutant and wt actins in the absence of regulatory
proteins were the same, as was also the binding of S1·ADP to actin-Tm
complexes containing either wt or mutant actin (Table I).
The strong binding interactions (i.e. in the absence of
nucleotides) between each of the actins and S1 were examined using cosedimentation assays. Here too, the results did not reveal any significant difference between the two actins in their binding of S1
(Table I).
Finally, we compared the pCa dependence of regulated acto-S1 Mg-ATPase
of both wt and 311/312 mutant actin to determine whether the mutation
alters the calcium affinity of TnC in the regulatory complex (Fig.
5). We found no difference between the
ATPases measured with wt or 311/312 mutant actin. Both had the same
degree of activation versus pCa and the same maximal ATPase
rates in experiments using bovine cardiac Tn and either bovine cardiac
Tm (Fig. 5) or skeletal Tm (data not shown). We thus conclude that
there is no difference in Ca2+ affinity between thin
filaments reconstituted with either of the actins.
All in all, the above assays suggest that there are no significant
structural differences between wt and the 311/312 mutant actin. The
results of the polymerization, acto-S1 MgATPase, regulated acto-S1
MgATPase, in vitro motility, S1 and S1·ADP binding, CD, and tryptophan fluorescence experiments strongly indicate that neither
the structure nor the function of the mutant actin has been modified.
Thus, the differences in the in vitro motility regulation
that we report in this study probably stem from the differences in the
interactions between the actins and the regulatory proteins.
The goal of this study was to test predictions of the Lorenz
et al. (1) model of the actin-Tm complex in the closed
state. According to this model, actin residue 311 contributes
significantly to the closed-state interaction of actin and Tm. Our
initial hypothesis, based on the above model, was that a mutation at
this residue should reduce the affinity of Tm for actin by about 4-fold
and thus destabilize the closed-state binding of Tm to the mutant actin. This, in turn, would be reflected in an altered Ca2+
sensitivity of the regulated mutant actin filaments in the in vitro motility assays.
As a first step, we established that wt yeast actin is fully regulated
by Tm-Tn (38). Thus, one important result of this work is that
regulation can be conveniently studied using yeast actin in the
in vitro motility assays. Korman and Tobacman (39) have also
shown yeast actin to be fully regulated in acto-S1 ATPase studies.
These results pave the way for regulation experiments using mutated
yeast actins.
The main result of this work is that in the in vitro
motility assay, the regulated 311/312 mutant actin filaments move
faster at high pCa values than do regulated wt actin filaments. Because structural and functional comparisons of the two actins did not reveal
any significant differences, these motility results indicate an
increased Ca2+ sensitivity of the regulated 311/312 actin filaments.
At first glance, these results appear consistent with the prediction of
Lorenz et al. (1) regarding the role of actin residue 311 in
actin-Tm binding. According to the McKillop and Geeves (4) three-state
model, the binding of Ca2+ to Tn induces conformational
changes and steric transitions, which expose myosin weak binding sites
on F-actin. At the same time the Tm-Tn complex moves into the closed
position. If, in the mutant actin thin filaments, the Tm-Tn complex is
not as firmly stabilized in the closed (+Ca2+) position,
then a smaller number of myosin heads will be sufficient to induce a
shift of the Tm-Tn complex into the open position. This should be
especially apparent at low Ca2+ concentrations, where few
cross-bridges bind to the thin filaments. Our findings seemed to
indicate that this is the case. In addition, the fact that larger
fractions of mutant than of wt filaments moved at high pCa values lent
support to this premise.
The explanation that the mutant actin regulation results are due, at
least to some extent, to the destabilization of the actin-Tm complex in
the closed state is not indicated by direct binding measurements. Tm
binds to wt and 311/312 mutant actins with similar affinities under
both closed-state (Tm alone) and open-state (Tm in the presence of S1
at a saturating concentration) conditions. Similar binding of S1·ADP
to wt and mutant actin-Tm complexes rules out another possible cause
for unequal stability of these actin-Tm complexes in the closed state.
The unchanged binding properties of the 311/312 mutant suggest that
explanations other than changes in equilibrium binding must be
considered to account for our observations. These possibilities
include:
The three-state equilibrium could be shifted toward the open state if
the mutaton actually increases the 311/312 actin open-state affinity
for Tm. However, our results suggest that Tm binding to actin under
open-state conditions is unchanged by the mutation. This implies that
the improved sliding of regulated 311/312 actin filaments at high pCa
is not caused by a stabilization of the open-state complex.
The increased Ca2+ sensitivity of the regulated 311/312
mutant actin may be because of a destabilization of the actin-Tm-Tn complex in the blocked state (+Tn, Regarding changes in blocked state stability, although we cannot
exclude this possibility, circumstantial evidence argues against it.
The modulation of actin motility by Tm alone shows that 311/312
mutation-induced changes also occur in the absence of the blocked
state. Moreover, because actin residues 311 and 312 are not within the
Tm binding site in the blocked state, any change in such binding to
311/312 mutant actin would be allosteric in nature.
Interpreting our data in terms of the Lorenz et al. (1)
model of the actin-Tm complex and the three-state model of regulation (4) may not be the only way to approach this issue. Squire and Morris
(42) speculate that Tm alone could occupy a range of positions on the
F-actin filament and that the modeled closed state of Tm on actin (1)
could very well be an average of these Tm positions. If so, this would
affect the identification of amino acid residues involved in the
actin-Tm interaction and the estimation of their Finally, the 311/312 mutation may modify the regulation via allosteric
shifts in the regulated actin system. One possible scenario is that the
binding of S1 to the actin-Tm-Tn complex in the presence of ATP and at
low calcium concentrations is enhanced by this mutation. Results of
motility experiments with the actin-Tm (no Tn) complexes are also
consistent with allosteric explanations. Inhibition of actin sliding by
Tm at low densities of HMM on the motility assay surface could be
described in purely steric terms, i.e. an insufficient
density of myosin heads binding to the closed-state actin-Tm complex to
tilt the equilibrium toward the open state (active form) of this
complex. (This argument may also explain the prior observations that
acto-S1 MgATPase is inhibited by Tm at low S1 concentrations (5-7).)
However, the potentiation of the 311/312 mutant actin-Tm sliding at
higher concentrations of HMM together with the reported potentiation of
acto-S1 MgATPase by Tm as S1 concentrations are increased (5-7)
(albeit not at conditions close to Vmax) cannot
be explained using a steric-block model of regulation or stronger
S1·ADP binding to the mutant actin-Tm complex. These findings point
to an allosteric change in the mutant actin-Tm complex leading to
kinetic and/or mechanical consequences in the actomyosin cross-bridge
cycle. Such consequences could, for example, include an increase in the
rate of the ADP release step and/or of the ATP binding step and the
subsequently more rapid release of the actin-Tm complex.
It should be noted that a missense mutation in actin (R312H) has been
associated with hereditary idiopathic dilated cardiomyopathy, a
heart failure of unknown origin (44). Nearly one-third of all heart
failures are associated with relaxation abnormalities that could stem
from an increased affinity of TnC for calcium, impaired sequestration
of calcium by the sarcoplasmic reticulum, or slowed extrusion of
calcium by the Na+/Ca2+ exchanger (45, 46). The
elevated pCa50 for the speed-pCa curve (Fig. 2A)
suggests that regulated thin filaments containing the E311A/R312A actin
mutant would exhibit slowed or impaired relaxation following
contraction. A similar effect occurring in thin filaments containing
the R312H mutation could contribute to the etiology of idiopathic
dilated cardiomyopathy.
In summary, using in vitro motility assays, we found that
yeast actin is a convenient substitute for skeletal actin in the study
of regulation. Our in vitro motility experiments uncovered notable differences in regulation between wt and 311/312 mutant actin.
As indicated by regulated acto-S1 ATPase assays, we saw no differences
in the affinity of Tn for Ca2+ in thin filaments
reconstituted with either actin. We detected no significant differences
between the two actins in their binding of Tm, S1, and S1·ADP and
small, if any differences in other nonregulated functions. Our results
underscore the importance of allosteric factors in the regulation of
actomyosin interactions.
*
This work was supported by United States Public Health
Service Grants AR22031 (to E. R.) and AR30988 (to E. H.) and National Science Foundation Grant MCB9630997 (to E. R.).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.
The abbreviations used are:
Tm, tropomyosin;
Tn, troponin;
S1, myosin subfragment 1;
DTT, dithiothreitol;
HMM, heavy
meromyosin;
TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid;
wt, wild type.
Role of Residues 311/312 in Actin-Tropomyosin Interaction
IN VITRO MOTILITY STUDY USING YEAST ACTIN MUTANT
E311A/R312A*
,¶
Department of Chemistry and Biochemistry,
the ¶ Molecular Biology Institute, and the
§ Department of Physiology, University of California,
Los Angeles, California 90095
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin, is fully regulated when complexed with
tropomyosin (Tm) and troponin (Tn). Structure-function comparisons of
the wt and E311A/R312A actins show no significant differences between
them, and the unregulated F-actins slide at similar speeds in the
in vitro motility assay. However, in the presence of Tm and
Tn, the mutation increases both the sliding speed and the number of
moving filaments at high pCa values, shifting the speed-pCa curve
nearly 0.5 pCa units to the left. Tm alone (no Tn) inhibits the
motilities of both actins at low heavy meromyosin densities but
potentiates only the motility of the mutant actin at high heavy
meromyosin densities. Actin-Tm binding measurements indicate no
significant difference between wt and E311A/R312A actin in Tm binding.
These results implicate allosteric effects in the regulation of
actomyosin function by tropomyosin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
G contributions to this interaction. Our choice of
residue 311 as a suitable starting point to test the predictions of
this model and to gain additional insight into Tm regulatory function
was based on two criteria. First, according to Lorenz et al.
(1) residue 311 has a relatively high energy contribution to actin-Tm
interaction, and secondly, there is a viable yeast actin mutant at this
location: E311A/R312A (20) (Fig. 1).
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Fig. 1.
F-actin monomer model of Lorenz et
al. (31) showing the locations of residues 311 (black) and 312 (gray) (displayed by
WebLab Viewer 2.0).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol were
purchased from Sigma. Yeast extract and tryptone were purchased from
Difco. DNase I was purchased from Roche Molecular Biochemicals.
-mercaptoethanol, pH 7.6). Skeletal tropomyosin was
prepared according to a previously reported protocol (26), as was
cardiac troponin (27). N-(1-pyrenyl)iodoacetamide-labeled Tm
was prepared using the protocol of Ishii and Lehrer (28) with a
pyrene-to-Tm labeling ratio of 2.0. The cardiac troponin and
tropomyosin were generous gifts from Dr. L. Tobacman. Yeast actin
strains DBY6962 and DBY6958 (20), producing the E311A/R312A and
K315A/E316A mutant actins, respectively, were generous gifts from Drs.
D. Botstein and T. C. Doyle.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
850 cal/mol). If this is
the case, replacing the charged glu-311 with an uncharged alanine could
partially destabilize the closed state, causing changes in the
regulation of the E311A/R312A actin mutant.
View larger version (20K):
[in a new window]
Fig. 2.
Effect of pCa on the in vitro
motility of regulated actin filaments. The speeds and
numbers of smoothly moving actin-Tm-Tn filaments were measured at
25 °C as they moved over a lawn of HMM adsorbed onto a
nitrocellulose-coated surface. The pCa of the motility assay buffer
(0.1 µM Tm, 0.1 µM Tn, 1 mM
ATP, 10 mM imidazole, pH 7.4, 25 mM KCl, 1 mM EGTA, 4.0 mM MgCl2, and 10 mM DTT) was varied from 7.5 to 5.0. Panel A, the
mean speeds of wt ( 
) and 311/312 mutant (
) actins are plotted
versus pCa. Error bars represent standard errors
for the mean speeds at each pCa value. The speeds of between 104 and
313 filaments were measured at each pCa in these experiments.
Panel B, percentages of wt (black) and 311/312
mutant (white) filaments moving at each of the
Ca2+ concentrations used.
Structure-function comparison of wt and 311/312 mutant actins
330 versus 850 cal/mol). Thus, any changes in the regulation
of this mutant actin should be smaller than those observed with
E311A/R312A actin. Indeed, the pCa titration results of the 315/316
mutant (both the sliding speeds and percentage of filaments moving)
fell between those of the other two actins (data not shown).
View larger version (18K):
[in a new window]
Fig. 3.
Effect of HMM surface density on the in
vitro motility of actin-Tm thin filaments. The density
of the HMM-coated surface was varied by applying HMM at concentrations
between 0.06 and 0.3 mg/ml to the nitrocellulose-covered surface of the
assay coverslip. Panel A, speeds of wt actin filaments in
the presence ( ) and absence () of Tm. Panel
B, speeds of 311/312 actin filaments in the presence
(
) and absence (
) of Tm. Between 52 and 174 filaments were
monitored at each HMM concentration in these experiments.
1 and (2.5 ± 0.4) × 106 M1 for wt and 311/312
actin, respectively. In terms of the Lorenz et al. model
(1), the predicted loss of binding energy contribution because of the
E311A replacement (850 cal/mol (1)) should result in an approximately
4-fold decrease in the Ka value of Tm for actin.
However, it should be noted that our studies were performed with the
double mutant E311A/R312A. Although residue Arg-312 was not credited
with having a significant contribution to the G of Tm
binding to actin (1), it may actually impact this interaction. Thus,
removing a positively charged residue (Arg-312) in addition to the
negatively charged Glu-311 could decrease or obviate the effect of the
311 substitution on actin-Tm binding. To test the possibility that the
mutant actin favors the binding of Tm in the open state, we also
measured Tm binding to actin in the presence of rigor bound S1. Here,
too, no significant differences between the 311/312 mutant and wt
actins were noted under these conditions (data not shown). Finally, we
checked whether the different effects of Tm on the sliding of wt and
mutant actins in the in vitro motility assays might be
related to different affinities of S1·ADP to their actin-Tm
complexes. Binding experiments did not reveal any significant
differences between the binding constants and Hill coefficients for
S1-ADP interactions with wt and mutant actin-Tm (Table I).
View larger version (17K):
[in a new window]
Fig. 4.
Binding of Tm to wt and 311/312 actins.
The actin-Tm cosedimentation binding assays were performed using wt
( ) and 311/312 mutant () actins (5.0 µM) and 0-2.2
µM N-(1-pyrenyl)iodoacetamide-labeled Tm
(PIATm) in a 5.0 mM HEPES buffer, pH 7.5, 3.0 mM MgCl2, 150 mM NaCl at 22 °C.
The data were fitted with sigmoidal curves using SigmaPlot 4.0 (Jandel
Scientific) and yielded Hill coefficients and binding constants of
2.4 ± 0.4, Ka = (3.2 ± 0.2) × 106 M1 for Tm binding to wt actin
and 2.1 ± 0.6, Ka = (2.5 ± 0.4) × 106 M1 for binding to the 311/312
mutant actin.
View larger version (12K):
[in a new window]
Fig. 5.
Activation of S1 ATPase activity by regulated
wt and 311/312 mutant versus pCa. The degree of
activation was monitored for thin filaments reconstituted with wt ( 
)
and 311/132 () actins by monitoring the clearing time of the assay
solutions (see "Materials and Methods"). The assays were performed
using 7.0 µM actin, 1.0 µM S1, 2.0 µM Tm, and 1.2 µM Tn in a buffer of 5.0 mM imidazole, pH 7.5, 3.0 mM MgCl2
(free), 13.2 mM KCl, 2.0 mM EGTA
(K2EGTA + Ca2+-K+-EGTA), 10 mM DTT, 0.1 mM ATP at 23 °C. The total ionic
strength was kept at 30 mM. Both actins produced the same
maximal S1 ATPase rates (1.5 ± 0.2 s1 at pCa 5) and
showed almost exactly the same courses of ATPase activation
versus pCa.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Ca2+), shifting the
equilibrium toward the closed state. Such an effect could be the result
of a reduced blocked-state affinity for Tm or an increase in the
affinity of Tn for Ca2+ in the mutant actin-Tm-Tn complex.
Using regulated actin-activated S1 MgATPase assays, we showed that the
311/312 mutation does not change the affinity of Tn for calcium. It may
appear surprising that the pCa profiles for regulated wt and mutant
actin filaments are the same in ATPase activity measurements but
different in the in vitro motility assays. However, the
rate-limiting steps in the two types of experiments are different.
Unlike the ATPase reaction, filament sliding is an analog of unloaded
muscle fiber shortening and is rate-limited by ADP release from
actomyosin·ADP (40). Consequently, ATPase values are not necessarily
predictive of filament sliding speeds in the motility assays (41).
G
contributions. An example of this would be the recent study of the
actin mutation E93K in the Drosophila flight muscle (43).
This residue was not implicated in the Lorenz et al. (1) study but, nevertheless, strongly affects the function of Actin-Tm filaments in the in vitro motility assay.
FOOTNOTES
To whom correspondence should be addressed. Tel.:
310-825-2668; Fax: 310-206-7286; E-mail: reisler{at}mbi.ucla.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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