From the
Earlier, we proposed that the interaction of gizzard calponin
with F-actin, promoting the inhibition of the actomyosin ATPase
activity, involves the NH
Smooth muscle calponin is an actin- and tropomyosin-binding
protein which may modulate at the thin filament level the actin-myosin
interactions and force generation
(1, 2, 3) .
Solution studies and in vitro motility assays have shown that
its binding to F-actin or F-actin-tropomyosin promotes the inhibition
of the actin-stimulated myosin ATPase
(4, 5, 6, 7) and actin sliding over myosin
(8, 9) . This
inhibitory activity can be reversed by complexation of calponin to a
calcium-dependent protein, like calmodulin, caltropin, or protein S100b
(10, 11) as well as by its site-specific
phosphorylation by protein kinase C or
Ca
To define the
molecular basis of calponin functions, we have earlier investigated the
distribution of its regulatory F-actin and calmodulin interactive sites
over three chymotryptic fragments isolated from gizzard calponin and
accounting for its entire primary structure
(18) . We found that
all the known biological activities of the protein were associated with
its NH
On-line formulae not verified for accuracy = 7.5, 11.7, 2.2, 2.0, 6.8, and 7.0
(18, 21) , respectively. The concentration of the
chymotryptic or synthetic peptides was determined from their absorbance
at 280 nm and calculated on the basis of their tryptophan content
(24) . The concentrations obtained were in good agreement with
those determined from the amino acid composition. The concentrations of
TY12, AF8, and QI11 were estimated by amino acid analysis.
Affinity chromatography of the peptides over
tropomyosin-Sepharose was performed in 10 m
M Tris-HCl, 10
m
M KCl, 1 m
M MgCl
Fluorescence emission spectra of the calmodulin-TW31
complex and of the complexes between pyrene-tropomyosin and synthetic
peptides were recorded as reported earlier
(20, 22) .
Effect of the Limited Chymotryptic Digestion of the Calponin
Fragment of Residues 7-182 on Its Inhibitory Activity-When
the isolated 22-kDa NH
Each peptide represented a single major component showing less than
2% impurity as assessed by reversed-phase HPLC and mass spectrometry
(Fig. 2). Amino acid composition analyses, sequence determinations, and
molecular masses were consistent with the expected sequences. All
peptides were readily soluble in aqueous buffer. We investigated
comparatively their binding to F-actin by co-sedimentation as well as
by EDC cross-linking in the absence or presence of
Ca
Following centrifugation of each peptide
in the absence or presence of F-actin (actin:peptide molar ratio, 1:8),
the corresponding pellets and supernatants were analyzed either by
electrophoresis on a 7.5-20% gradient acrylamide gel or by
reverse-phase HPLC. As illustrated in Fig. 3, TW31 and AW19
displayed significant binding to F-actin, and no pelleting of either
peptide occurred without actin. The peptide analogs including
isoleucine instead of tryptophan at the COOH terminus behaved
essentially similarly (data not shown). Because TY12 (Fig. 3) as
well as AF8 and QI11 were small-sized peptides, their respective pellet
and supernatant fractions could not be analyzed by gel electrophoresis
and were subjected to reverse-phase HPLC. No trace of either peptide
was found associated with F-actin (data not shown). The actin-binding
peptides TW31 and AW19 share a common sequence, spanned by
Ala
In this study, we have used synthetic peptides encompassing
the calponin sequence Thr
Our
investigations revealed also another striking function associated
mainly with the 153-163 segment and consisting in its
complexation with tropomyosin. Calponin was earlier shown to bind to
the tropomyosin sequence of residues 142-227
(29) , but
the tropomyosin-binding site(s) on calponin has not yet been precisely
delineated. The calponin 153-163 stretch is certainly not the
only tropomyosin binding site of calponin since interaction between
tropomyosin and the calponin segment of residues 7-144 was also
demonstrated
(26) . To account for the occurrence of at least
two classes of tropomyosin binding regions on calponin, we have
referred, in Fig. 10 A, to the NH
The amino acid sequence of the multifunctional 145-163 region
is highly conserved in all the known smooth muscle calponin isoforms,
suggesting that it is important for the biological activity of the
calponin molecule. Recently, a novel calponin variant was identified in
rat aorta and non-muscle cells such as the central brain, and its
covalent structure was established
(37) . It differs from the
other calponins by the inclusion of extensive changes in the
NH
Finally, the
145-163 region we have characterized exhibits reciprocal
structural and functional relationships with the adjacent residues of
Ser
Each peptide was submitted to
calmodulin-Sepharose or tropomyosin-Sepharose affinity chromatography
as described under ``Experimental Procedures''; the sign
(+) means that the peptide exhibits reversible binding to the
column whereas the sign (-) indicates that the peptide elutes in
the flow-through fraction.
We thank Dr. Mario Gimona for communicating
prepublication data on the interaction of recombinant calponin
fragments with calmodulin and caltropin. We also thank Dr. J. P. Capony
for amino acid analyses.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-termi-nal portion of the calponin
segment Ala
-Tyr
(Mezgueldi, M., Fattoum,
A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267,
15943-15951). In this work, we have directly probed this region
for actin binding sites using five peptide analogs covering different
stretches of the sequence Thr
-Ile
.
Co-sedimentation with F-actin, actomyosin ATPase measurements, and
zero-length cross-linking reactions demonstrated that the 19-residue
sequence Ala
-Ile
is essential for actin
interaction and ATPase inhibition. Furthermore, each peptide was tested
for binding to the Ca
-dependent proteins, caltropin
and calmodulin, in both an actomyosin ATPase assay and an affinity
chromatographic assay. The results revealed the 11-residue segment
Gln
-Ile
, representing the COOH-terminal
moiety of the F-actin binding sequence, as a crucial region for the
high affinity binding of these regulatory proteins with concomitant
removal of the ATPase inhibition. The 153-163 stretch contained
also interactive sites for tropomyosin as assessed by affinity
chromatography and spectrofluorometry. Collectively, the data support
our initial results and highlight the ability of the multifunctional
145-163 region to serve as a potent regulatory domain of the
smooth muscle calponin.
-calmodulin-dependent protein kinase II
(4, 12, 13) . The identification of a calponin
phosphatase has suggested that a cycle of
phosphorylation-dephosphorylation of the protein may regulate the
cross-bridge activity in vivo (4, 14, 15, 16) . The recently
described occurrence of calponin in the actomyosin domain of the smooth
muscle cell further strengthens its proposed involvement in the
regulation of smooth muscle contraction
(17) .
-terminal segment spanned by residues 7-182
whereas no detectable function was displayed by the remaining
COOH-terminal 183-292 region. Essentially similar observations
were subsequently reported by others
(13) . Most importantly,
our studies, using proteolytic digestions and zero-length cross-linking
of the F-actin-calponin complex, strongly suggested but did not prove
that the inhibitory binding of actin takes place on the 38-residue
stretch between Ala
and Tyr
, the most
exposed and protease-sensitive region on the surface of calponin
(3) . Residues present toward the amino-terminal end of this
sequence have been proposed to interact with actin. Our data also led
to the conclusion that the segment spanning amino acids 61-182
includes major determinants for the recognition of F-actin, calmodulin,
and tropomyosin. More recently, the binding stoichiometry for the
complex of calponin and calmodulin or caltropin was determined to be
1:2 with the interaction of either calcium-dependent protein at a low
and at a high affinity site on the calponin molecule
(10, 11) . The existence of the two different sites was
confirmed by briefly described titration analyses of the binding of the
former proteins to a recombinant fragment of calponin spanning residues
1-228
(19) ; these data further indicated that the weaker
binding site was within the NH
-terminal segment 2-51
and the stronger binding site was residing between residues 52 and 228,
a region which also comprises our putative inhibitory F-actin
interactive domain. However, the functional and structural
relationships between the latter crucial site and the pair of sites
involved in the recognition of the calcium-dependent proteins remain to
be elucidated. In the present work, we have assessed the functional
coupling between the actin site and the site of tight binding of the
calcium-dependent proteins, by determining more precisely the relative
location of their respective critical determinants within the covalent
structure of calponin. Taking advantage of our recent findings with
caldesmon revealing the juxtaposition of the regulatory F-actin and
calmodulin binding sequences on the primary structure of its
COOH-terminal domain
(20) , we have synthesized a series of
oligopeptides corresponding to different stretches of the calponin
amino acid sequence from Thr
to Ile
. This
segment encompasses the NH
-terminal moiety of our proposed
F-actin binding region of residues 145-182
(18) . We have
probed in detail their biological properties using F-actin
co-sedimentation, actomyosin-S-1 ATPase assays, cross-linking reactions
with F-actin or calmodulin, and affinity chromatography over
immobilized calmodulin or tropomyosin. The data demonstrate for the
first time the direct interaction of the 19-residue segment
Ala
-Ile
with F-actin,
Ca
-calmodulin, Ca
-caltropin, and
tropomyosin. Its complexation to actin occurs competitively with the
binding of the functional calponin fragment of residues 7-182 and
causes the inhibition of ATPase activation in a calmodulin- or
caltropin-regulated manner, whereas its association with tropomyosin
was without consequences. The interaction of the calcium-dependent
proteins and tropomyosin was restricted to the COOH-terminal portion
Gln
-Ile
; in contrast, actin recognition
required the entire 145-163 segment. These studies highlight
several interaction properties of calponin and provide the experimental
evidence for the previously assigned location of its inhibitory actin
binding site
(18) . They also suggest that the multifunctional
features of the 145-163 sequence combined with its close
proximity in calponin primary structure to the phosphorylatable
residues, Ser
/Thr
, are likely reflecting
the ability of this particular region to act as a potent regulatory
domain of the native protein.
Materials
-Chymotrypsin was purchased from
Worthington Biochemical Corp. Calmodulin-Sepharose and CNBr-activated
Sepharose 4B were from Pharmacia Biotech Inc. The latter material was
coupled to tropomyosin according to the manufacturer's
specifications. Fmoc amino acids were obtained from Milligen
(Millipore). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC)
(
)
and N-hydroxysuccinimide (NHS)
were from Sigma and Serva, Heidelberg, respectively.
Proteins and Peptides
Calponin and tropomyosin
from fresh turkey gizzards, F-actin, and chymotryptic myosin
subfragment-1 (S-1) from rabbit skeletal muscle were prepared as
described previously
(18) . Bovine brain calmodulin and chicken
gizzard caltropin were purified by published procedures
(20, 21) . Tropomyosin was labeled at Cyswith pyrene- N-maleimide as reported earlier
(22) . The 22-kDa and 13-kDa NH
-terminal
chymotryptic fragments of calponin (CH22 and CH13N, respectively) were
produced according to Mezgueldi et al. (18) and were
purified over immobilized tropomyosin. Synthetic peptides were prepared
and purified as reported earlier
(20, 23) . They were
subjected to amino acid analysis, sequence determination, and molecular
mass measurement using electrospray ionization mass spectrometry.
Protein Concentrations
The concentrations of S-1,
F-actin, tropomyosin, calmodulin, caltropin, and calponin were measured
spectrophotometrically using
Gel Electrophoresis
SDS-polyacrylamide gel
electrophoresis was carried out in 3-18% or 7.5-20%
gradient acrylamide gels
(25) . The running buffer was 50 m
M Tris, 100 m
M boric acid, pH 8.0. Marker peptides used
were myoglobin CNBr fragments: 2.6 kDa, 6.4 kDa, 14.6 kDa, and 17.2
kDa. The immunoblotting with polyclonal actin antibodies was performed
as reported earlier
(18) .
F-actin Binding Assays
The interaction of F-actin
and peptides was analyzed by co-sedimentation in 10 m
M imidazole-HCl, 30 m
M NaCl, 2 m
M MgCl, 1 m
M NaN
, pH 7.0, using a
Beckman Airfuge at 140,000
g, for 30 min at 25 °C.
The pellets were homogenized in the same buffer and analyzed either
qualitatively by gel electrophoresis or quantitatively by HPLC on a
reverse-phase Aquapore C-8 column (4 mm
250 mm) eluted with
0-60% acetonitrile gradient containing 0.1% trifluoroacetic acid
(20) .
Chymotryptic Digestions
Calponin or its 22-kDa
fragment was treated with chymotrypsin at 25 °C using a protease to
substrate weight ratio of 1:100, in 10 m
M imidazole HCl, 30
m
M NaCl, 2 m
M MgCl, 1 m
M NaN
, pH 7.0. At the desired time intervals (0-60
min), protein aliquots were analyzed by gel electrophoresis and assayed
for inhibition of the acto-S-1 ATPase after the addition of 2.5 m
M phenylmethylsulfonyl fluoride.
Cross-linking Reactions
TW31 (0.15 mg/ml) in 10
m
M imidazole HCl, 30 m
M NaCl, 2 m
M MgCl, 1 m
M NaN
, pH. 7.0, was
mixed with F-actin (0.21 mg/ml) (TW31:actin molar ratio = 8:1)
in the presence of 2 m
M EDC and 5 m
M NHS. The
reaction was carried out at 25 °C for 0-30 min in the absence
or presence of Ca
-calmodulin (5.5 mg/ml)
(TW31:Ca
-calmodulin molar ratio = 1:8) and was
monitored by SDS-gel electrophoresis.
Calmodulin and Tropomyosin Binding
Measurements
The binding of peptides to calmodulin was assayed
by calmodulin-Sepharose affinity chromatography as described previously
(20) .
, 0.1 m
M dithiothreitol, and 1 m
M NaN
, pH 7.0. Bound
peptides were eluted with the same buffer containing 200 m
M KCl.
ATPase Measurements
The
Mg-ATPase of the skeletal acto-S-1 was determined in
a medium (1 ml) containing 50 m
M Tris-HCl, 5 m
M ATP,
10 m
M KCl, 2.5 m
M MgCl
, pH 7.5, using
0.42 mg/ml F-actin and 0.050 mg/ml S-1. P
was determined
colorimetrically as described previously
(18) .
-terminal chymotryptic fragment of
calponin spanning amino acids 7-182 was further incubated with
chymotrypsin at a protease to substrate weight ratio of 1:100, it was
rapidly and quantitatively converted into the stable peptide designated
CH13N (Fig. 1 A). Earlier, this component was shown to
derive from the cleavage, under identical conditions, of the intact
calponin at Tyr
-Ala
and was identified as
the NH
-terminal segment of residues 7-144
(18, 26) . Thus, the direct proteolysis of the 22-kDa
fragment did result only in the excision of its COOH-terminal
38-residue stretch, between Ala
and Tyr
,
thought to contain the inhibitory actin-binding region
(18) .
Most interestingly, when the whole digest of the 22-kDa peptide was
subjected in parallel to acto-S-1 ATPase assays during the entire
course of the proteolytic process, no change in the inhibitory
capability of the peptide was noticed even after a 60-min reaction
(Fig. 1 B). The digestion, under the same experimental
conditions, of the parent calponin, used as control, also failed to
alter significantly the extent of ATPase inhibition. The initial 70%
inhibition of the activity tended to level off to the same value
(
60%) as that found for the digested 22-kDa fragment
(Fig. 1 B). These observations strongly suggest that the
proteolysis had no deleterious effect on the major structural
determinants for specific binding to F-actin that are still operant in
the released 38-residue peptide or in its chymotryptic subfragments.
There is no doubt that only the presence of the latter fragment(s) in
the digest solution is responsible for the observed maintenance of the
ATPase inhibition since, as indicated in Fig. 1 C, only
the intact 22-kDa fragment but not its isolated 13-kDa
NH
-terminal portion could inhibit the acto-S-1 ATPase in a
concentration-dependent manner. The inefficiency of the 13-kDa fragment
was not caused by its isolation since, earlier, we have shown that this
material did not co-sediment with F-actin directly added to the total
digest of calponin
(18) . Also, all of the 13-kDa peptide
remained in the supernatant upon centrifugation of a mixture of F-actin
and the 60-min digest of the 22-kDa peptide (data not shown).
Figure 1:
Chymotryptic digestion of the CH22
fragment and its consequence on the acto-S-1 ATPase inhibition by the
fragment. A, CH22 fragment (2.0 mg/ml) in 10 m
M imidazole HCl, 30 m
M NaCl, 2 m
M MgCl, 1 m
M NaN
, pH 7.0, was
digested with
-chymotrypsin at 25 °C (protease to substrate
weight ratio = 1:100). At the times indicated, the digest was
analyzed by electrophoresis on 7.5-20% gradient acrylamide gel.
B, at the indicated time intervals, aliquots of the digest of
CH22 (
) were subjected to acto-S-1 ATPase assays. Aliquots of a
chymotryptic digest of calponin (
), produced under the conditions
reported in A, were similarly tested for inhibition of the
acto-S-1 ATPase activity (actin:calponin or CH22 molar ratio =
1:0.5). C, acto-S-1 ATPase assays were carried out using
increasing concentrations of the isolated fragments CH22 (
) or
CH13N (
). 100% ATPase = 0.450 µmol of
P
/min/mg.
The
apparent conservation of the inhibitory function in the proteolytically
detached 145-182 region encouraged us to prepare various
synthetic peptides covering the NH-terminal sequence
145-163 as probes of the interaction between actin and this
region.
Ca
The peptides we have
synthesized corresponded to different stretches of the main sequence
Thr-Calmodulin-regulated Interaction of
F-actin with the Synthetic Peptides Including the Sequence
Ala
-Ile
-Ile
. In some peptides, Ile
was replaced by a tryptophan residue. This amino acid conferred
characteristic spectral features without influencing the biological
activities of the peptide as verified by comparing the functional
properties of the peptide analogs comprising either isoleucine or
tryptophan at position 163. The synthetic peptides used in this study
were designated TW31 (spanning the 31-residue sequence
Thr
-Trp
), TY12 (spanning the 12-residue
sequence Thr
-Tyr
), AW19 (spanning the
19-residue sequence Ala
-Trp
), AF8 (spanning
the 8-residue sequence Ala
-Phe
), and QI11
(spanning the 11-residue sequence Gln
-Ile
).
-calmodulin. We also tested their ability to
inhibit the acto-S-1 ATPase and the influence of the
Ca
-dependent proteins, caltropin and calmodulin, on
their inhibitory activity.
-Ile(Trp)
, which must include the
critical determinants for F-actin binding; this proposal is consistent
with the absence of F-actin binding to the difference peptide TY12. On
the other hand, the lack of actin interaction with its separated
NH
-terminal and COOH-terminal moieties, represented by AF8
and QI11, respectively, strongly suggests that the structural integrity
of the full-length 19-residue sequence between Ala
and
Ile
is required for F-actin recognition.
Figure 3:
Interaction between F-actin and the
synthetic peptides. The binding of the synthetic peptides, TW31, TY12,
and AW19, to F-actin (actin:peptide molar ratio = 1:8) was
analyzed by co-sedimentation at 140,000 g for 30 min
at 25 °C followed by electrophoresis of the supernatant and pellet
fractions on a 7.5-20% gradient acrylamide gel. The binding of
calponin ( CaP) and CH22 to F-actin was assayed similarly as a
control. Lanes a and b, supernatant and pellet,
respectively, of each peptide alone. Lanes c and d,
supernatant and pellet, respectively, of each peptide centrifuged with
F-actin.
To assess
that the latter critical sequence also significantly contributes to the
complexation of the functional 22-kDa chymotryptic fragment of calponin
(residues 7-182) to F-actin, we investigated the competition
between this fragment and TW31 for binding to F-actin. Fig. 4, A and B, shows the potency of TW31 to displace the
proteolytic fragment from actin in a concentration-dependent manner.
About 60% of the bound fragment was dissociated from actin at a
TW31:CH22 molar ratio near 10. This finding indicates that TW31
competes with CH22 for a common binding site on F-actin. Previously, we
have shown that native calponin or CH22 could be specifically
cross-linked to F-actin by EDC, and we identified the cross-linked site
on the actin carboxyl-terminal region between residues 326 and 355
(18) . The actin binding activity observed for the synthetic
TW31 led us to analyze its EDC cross-linking to F-actin. The time
course of the reaction is depicted in Fig. 5 A, lane
c, using a molar ratio of actin to peptide of 1:8. A new major
species of 45 kDa, migrating just above the actin monomer, was
generated and its yield increased with the reaction time. It was
immunostained with the actin antibodies (Fig. 5 B). The 45-kDa
derivative likely represents a 1:1 molar covalent complex of actin and
TW31. Its production strongly suggests that the zero-length
cross-linking of either calponin or CH22 to F-actin reported earlier
(18) was mediated by the active sequence
Ala-Ile
. Significantly, Fig. 5 A,
lane g, shows that the formation of the 45-kDa adduct is
almost completely suppressed in the presence of
Ca
-calmodulin added at an 8-fold molar excess over
the synthetic peptide. Instead, a new entity of 21 kDa, with a mobility
slightly lower than calmodulin, was progressively produced. The same
species was also detected, under the same conditions, by reacting a
Ca
-calmodulin and TW31 mixture with the cross-linking
reagents (Fig. 5 A, lane e). The 21-kDa band did
not appear in the presence of EGTA (data not shown) and when
Ca
-calmodulin or TW31 was separately treated with EDC
(Fig. 5, lanes d and b, respectively).
Furthermore, the preformed 21-kDa material failed to co-sediment with
added F-actin both in the absence or presence of calcium (Fig.
5 C). These results are consistent with the idea that the
21-kDa material is resulting from a specific conjugation between TW31
and Ca
-calmodulin whose reversible or permanent
attachment to the peptide interferes with F-actin recognition. This
conclusion implies that the sequence Ala
-Ile
harbors interactive sites not only for F-actin but also for
Ca
-calmodulin.
Figure 5:
EDC cross-linking of the F-actin-TW31 and
Ca-calmodulin-TW31 complexes. A, F-actin and
TW31 mixed at a 1:8 molar ratio were covalently coupled with EDC-NHS as
described under ``Experimental Procedures.'' At the times
indicated, the cross-linking reaction was analyzed by 3-18%
gradient acrylamide gel electrophoresis in the absence ( lane
c) or presence ( lane g) of
Ca
-calmodulin. Lanes a, b, and
d, EDC-NHS-treated F-actin, TW31, and
Ca
-calmodulin, respectively. Lane e, EDC
cross-linking of the Ca
-calmodulin-TW31 complex.
Lane f, control EDC-treated
F-actin-Ca
-calmodulin mixture. The 45-kDa and 21-kDa
bands represent the 1:1 molar covalent complexes of TW31-actin and
TW31-calmodulin, respectively. St = protein markers.
B, the electrophoretic pattern of the F-actin-TW31
cross-linking reaction was immunostained with actin antibodies.
C, an aliquot of the 30-min cross-linking reaction mixture
including Ca
-calmodulin and TW31 was supplemented
with F-actin (molar ratio actin:TW31 = 1:8) in the absence
( lanes a and b) or presence ( lanes c and
d) of 5 m
M EGTA and centrifuged for 30 min at 140,000
g. The supernatants ( lanes a and c)
and the pellets ( lanes b and d) were examined by gel
electrophoresis.
Inactivation of the Acto-S-1 ATPase by the Peptide
Analogs of the Sequence Ala
As depicted in Fig. 6 A, TW31 as
well as AW19 inhibited significantly the skeletal acto-S-1 ATPase
activity in a dose-dependent manner. The presence of tropomyosin in the
ATPase assays did not affect their inhibitory potency. In contrast, the
three other peptides, TY12, AF18, and QI11, which were unable to
co-sediment with F-actin, were inefficient. When the inhibition of
ATPase by TW31 was related to the amount of peptide bound to actin, it
was found that at least 70% inhibition was reached with about 1 mol of
peptide bound/mol of actin (Fig. 6 B). Thus, the actin
binding sequence Ala-Ile
and
Its Restoration by the Ca
-dependent
Proteins
-Ile
also promotes the
ATPase inhibition. Moreover, Fig. 7shows that the addition of
Ca
-calmodulin or Ca
-caltropin leads
to the reversal of a large extent of the peptide inhibition. In the
absence of calcium, no effect was observed at any concentration of
either protein (data not shown). As previously reported for the
inhibition by native calponin
(11) , caltropin was at least
twice as efficient as calmodulin in restoring the acto-S-1 ATPase
inhibited by AW19. The data clearly indicate that the interaction of
the Ca
-binding proteins with the stretch
Ala
-Ile
is functionally coupled with the
binding of F-actin to adjacent or overlapping sites within the same
region.
Figure 6:
Inhibition of the acto-S-1 ATPase activity
by the synthetic peptides TW31 and AW19. A, ATPase assays were
carried out, as specified under ``Experimental Procedures,''
using increasing concentrations of TW31 (), AW19 (
),
TY12 (
), QI11 (
), or AF8 (
). For TW31, the acto-S-1
ATPase was also measured in the presence of tropomyosin (
)
(actin:tropomyosin molar ratio = 7:1). 100% ATPase =
0.480 µmol of P
/min/mg. B, relationship
between the degree of ATPase inhibition and the quantity of TW31 bound
to F-actin. Parallel determinations were made of the ATPase rate and
the amount of peptide bound to F-actin after co-sedimentation and
analysis of the resulting pellet by quantitative reverse-phase HPLC on
a C-8 column as described under ``Experimental
Procedures.''
Figure 7:
Reversal of the AW19 inhibition of the
acto-S-1 ATPase activity by Ca-binding proteins.
ATPase assays were carried out as described under ``Experimental
Procedures'' using an 8-fold molar excess of the synthetic peptide
AW19 over F-actin in the presence of 1 m
M CaCl
and
increasing concentrations of calmodulin (
) or caltropin
(
). 100% ATPase = 0.490 µmol of
P
/min/mg.
Specific Binding of Ca
To further define the
minimal sequence necessary for full binding of the
Ca-Calmodulin
and Tropomyosin to the Sequence
Gln
-Ile
-dependent proteins, we subjected each of the five
synthetic peptides to calmodulin-Sepharose affinity chromatography. The
experiments were initiated in the presence of 1 m
M CaCl
and under the high ionic strength conditions (0.1
M KCl)
we previously employed to assess the interaction of the chymotryptic
CH22 fragment with immobilized calmodulin
(18) . The overall
data are presented in . Only the peptides TW31, AW19, and
QI11, which comprise the sequence
Gln
-Ile(Trp)
, displayed the capacity to
bind to the calmodulin column in a calcium-dependent manner, whereas
TY12 and AF8 were not at all retained. The elution of the bound
peptides was achieved only in the presence of EGTA and 0.2
M KCl (Fig. 8, A and B), essentially as
observed earlier for calponin or the CH22 fragment
(18) . These
results are in agreement with the above described ability of the
Ca
-binding proteins to modulate the inhibitory
activity of TW31 and AW19. Consequently, the caltropin and calmodulin
binding domain on the critical actin interactive sequence
Ala
-Ile
is confined to its COOH-terminal
11-residue segment between Gln
and Ile
. The
spectrofluorometric analysis of the complex between TW31 and
Ca
-calmodulin did not reveal any change in the
tryptophan fluorescence intensity (data not shown).
Figure 8:
Binding of the synthetic peptides TW31 and
QI11 to calmodulin-Sepharose. The peptides TW31 (0.3 mg) and QI11 (0.1
mg) in 10 m
M Tris HCl, 2 m
M MgCl, 0.1
m
M dithiothreitol, 100 m
M KCl, and 1 m
M CaCl
, pH 7.0, were loaded separately onto a
Ca
-calmodulin-Sepharose affinity column as specified
under ``Experimental Procedures.'' After washing with the
same buffer, the column was eluted with 200 m
M KCl and 1
m
M EGTA added at the point indicated by the arrow.
TW31 ( A) and QI11 ( B) were recovered only in the
retained fraction eluted by the EGTA-containing
buffer.
Because
Ca-calmodulin was reported to disrupt the association
between calponin and tropomyosin
(27, 28) , we also
tested in parallel the interaction of the synthesized peptides with
immobilized tropomyosin using neutral pH and 10 m
M KCl.
Interestingly, the data summarized in show that the
Ca
-calmodulin-binding peptides, TW31, AW19, and QI11
but not TY12 or AF8, were also completely retained by the
tropomyosin-affinity column. As illustrated in Fig. 9, A and B, either bound peptide could be eluted by raising
the salt concentration of the buffer above 0.1
M. The latter
experimental condition was shown earlier to promote the dissociation of
native calponin from immobilized tropomyosin
(26) . The findings
indicate that the sequence Gln
-Ile
may also
represent a potent tropomyosin binding region. To reinforce this
proposal, we compared the relative solution binding of tropomyosin to
the different peptides and to native calponin. Because, like caldesmon,
calponin interacts near cysteine 190 of tropomyosin
(29) , the
binding reactions were assayed by measuring the fluorescence changes of
pyrene-tropomyosin employed at a constant concentration, at 5 m
M MgCl
and 40 m
M KCl (Fig. 9 C).
Calponin enhanced the fluorescence by up to 22%, and the change
saturated at a calponin:tropomyosin molar ratio of around 3:1; a 35%
fluorescence increase was reported for caldesmon under identical
experimental conditions
(30) . Increasing concentrations of AI19
also enhanced fluorescence by about 70% as much as intact calponin. The
maximum fluorescence increase with QI11 was slightly lower accounting
for nearly 60% of the calponin effect, suggesting a weaker binding than
the longer AI19. In contrast, and as expected, no fluorescence change
was noticed on adding TY12. These results indicate that, at the
moderate salt concentration used, the changes in fluorescence of
pyrene-tropomyosin were not nonspecific but rather due to an
interaction between this protein and calponin or the peptide analogs
that were found to be retained on immobilized tropomyosin. The
influence of the latter peptides on the pyrene environment suggests
that, like the parent calponin, they did bind to the region of
tropomyosin including Cys
.
Figure 9:
Interaction of the synthetic peptides TW31
and QI11 with tropomyosin. The peptides TW31 (0.2 mg) and QI11 (0.1 mg)
in 10 m
M Tris HCl, 10 m
M KCl, 1 m
M MgCl, 0.1 m
M dithiothreitol, and 1 m
M NaN
, pH 7.0, were loaded separately onto a
tropomyosin-Sepharose column as described under ``Experimental
Procedures.'' The column was first washed with the same buffer and
then eluted with 200 m
M KCl added at the point indicated by
the arrow. Both peptides TW31 ( A) and QI11
( B) bound to the immobilized tropomyosin and were recovered
only in the fraction eluted by the 200 m
M KCl-containing
buffer. C, 0.5 µ
M tropomyosin labeled with
N-(1-pyrenyl)maleimide was mixed with 0-3 µ
M calponin (
) or calponin peptides AI19 (X), QI11 (
), or
TY 12 (
) in 5 m
M HEPES, 40 m
M KCl, 5 m
M MgCl
, 1 m
M dithiothreitol, pH 7.0, at 25
°C, and the fluorescence emission at 377 nm was measured after
excitation at 340 nm. Data are expressed as percent increase, as
compared with pyrene-tropomyosin alone.
-Ile
to
demonstrate the direct interaction of F-actin with the 19-residue
polypeptide chain between Ala
and Ile
that
we have earlier assumed to potentially represent the actin binding
region of the protein
(18) . Two peptide analogs comprising this
critical sequence (TW31 and AW19) bound reversibly as well as
covalently to F-actin and inhibited the acto-S-1 ATPase activity. In
this respect, they accurately mimicked the inhibitory patterns of
calponin, CH22, and the active subfragments produced in the limited
chymotryptic digest of either of the two latter proteins. The
similarity between the synthetic peptide pair and their parent proteins
is further underlined by the additional modulation of the inhibitory
function of the peptides consequent on their binding to the
calcium-dependent proteins, caltropin and calmodulin. We could
unambiguously identify the COOH-terminal 11-residue sequence
Gln
-Ile
as an essential stretch for the
recognition of these regulatory proteins and the removal of the ATPase
inhibition. Most probably, the same region is also serving for the
binding of the S100 protein
(10, 11) . In the diagram
presented in Fig. 10, we designated this 153-163 region as
the high affinity caltropin/calmodulin binding site 2 in order to
differentiate it from the lower affinity binding site 1 previously
localized in the NH
-terminal segment of residues 1-52
(19) . The 153-163 peptide would harbor the high affinity
site tentatively localized on the segment 52-228
(19) .
The juxtaposition and, possibly, the overlapping between CaT/CaM site 2
and the actin binding site within the overall sequence
Ala
-Ile
rationalize the ability of the
former site to mediate the restoration of the actomyosin ATPase upon
binding to the Ca
-dependent proteins. In this regard,
calponin resembles caldesmon since both proteins include two binding
sites for the Ca
-dependent proteins with the higher
affinity site being located in the primary structure adjacent to and
functionally coupled with an actin binding and inhibitory sequence
(20, 31) .
Figure 10:
A, diagram illustrating the localization
of essential structural determinants which potentially make up the
regulatory domain of smooth muscle calponin. These include the
identified 145-163 actin-binding and inhibitory segment, the
153-163 stretch representing the tight binding site of the
Ca-dependent proteins (caltropin (CaT) or calmodulin
(CaM)), and one of the recognition sites of tropomyosin (TM) together
with the adjacent Ser
/Thr
, the
phosphorylation of which modulates the calponin-actin interactions (4,
12, 13). The NH
-terminal 1-52 sequence houses the low
affinity binding site for the Ca
-dependent proteins
(19) and possibly another tropomyosin interactive region. B,
comparison of the amino acid sequences of gizzard ( a) and
acidic ( b) calponins in the regulatory 145-163 segment.
The sequences are those given for chicken gizzard (3) and rat aorta
(37) calponins, respectively.
The functional contribution of the
145-163 sequence is in agreement with our earlier findings
describing the cross-linking of actin to the calponin CNBr fragment of
residues 52-168
(18) . The inability of AF8 and QI11,
which together cover the 145-163 region, to associate separately
with actin can be explained by the fact that either a specific actin
binding conformation is conferred only by the entire 19-residue-long
sequence or that multiple, evenly distributed functional determinants
cooperatively interact with actin. The latter possibility would make
understandable the moderate loss of ATPase inhibition reported for a
calponin recombinant mutated at Lys(32) . On the
other hand, recent functional studies on mutant calponin fragments,
modified at residues 145-147, revealed the critical importance of
these three amino acids for ATPase inhibition.
(
)
The latter investigations further substantiate the
functional role of the 145-163 sequence in the calponin-actin
interactions. Furthermore, the double replacement of Gln
and Gln
by arginine had no noticeable consequences
as we assessed by analyzing a synthetic peptide analog containing these
substitutions. In contrast, we observed that the mild treatment of CH22
with the arginine-directed protease, clostripain, leads to the
production of the NH
-terminal 13-kDa peptide with a severe
loss of ATPase inhibition by the digest. This suggests that the
structural integrity of the arginyl peptide bonds within the inhibitory
145-163 region is essential for the maintenance of its function.
Consequently, the conservation of ATPase inhibition we found with the
chymotryptic digest of CH22 should imply the complete lack of
proteolytic cleavage of the latter active sequence at the potentially
susceptible peptide bond Phe
-Gln
. This
conclusion is consistent with the loss of the inhibitory function we
observed when using AF8 and QI11 separately. The higher concentrations
of the synthetic 145-163 peptide needed for ATPase inhibition
(Fig. 6), as compared to the chymotryptic 145-182 subfragment
(Fig. 1), does not reflect a greater inhibitory potency of the
latter material since both peptides inhibited the ATPase to the same
extent. This quantitative difference most likely results from a
difference in the affinity of the two sequences for actin. This
proposal is supported by the following two observations. First, a
K
of approximately 168 µ
M was determined for the complex of actin and the synthetic peptide,
indicating an affinity about 100-fold weaker than that of calponin.
Second, the investigations on the calponin mutants
showed
that the binding to actin is tighter when the sequence extends through
position 182. The recombinant fragment of residues 131-228 was
found to display ATPase inhibition with affinity for actin similar to
intact calponin. Our conclusions are in agreement with the briefly
reported functional properties of a synthetic calponin peptide spanning
residues 144-175 which inhibits the acto-S-1 ATPase with a much
weaker affinity for actin than calponin
(34) .
-terminally
located site as TM binding site 1 and to the 153-163 sequence as
TM binding site 2. These two sites do not exclude the interaction of
tropomyosin on other additional or complementary stretches. Their
juxtaposition to CaT/CaM site 1 and CaT/CaM site 2, respectively, would
make tropomyosin and the Ca
-dependent proteins
compete for binding to calponin. This proposal is consistent with the
reported property of Ca
-calmodulin or S100 protein to
dissociate the calponin-tropomyosin complex
(27, 28, 35) . On the other hand, the binding of
F-actin to the crucial sequence Ala
-Ile
could actually block the interaction of tropomyosin with its
binding site 2, thus explaining the absence of tropomyosin effect on
the inhibitory capacity of either calponin
(36) or the
synthetic peptides. So far, it remains to be elucidated whether
calponin could firmly attach to tropomyosin in the presence of F-actin.
-terminal third of the sequence and the addition of a
57-residue acidic domain at the COOH-terminal end. Significantly, this
protein was found unable to interact with immobilized calmodulin, a
behavior suggesting it has lost the pair of binding sites for the
Ca
-dependent proteins. While the alterations of the
sequence 1-52 may account for the suppression of CaT/CaM binding
site 1, Fig. 10 B clearly shows that 6 nonconservative
amino acid substitutions are also located on the 153-163 stretch
constituting CaT/CaM binding site 2 and are likely responsible for the
abolition of its interaction with calmodulin. It is noteworthy that the
latter structural changes did not affect the hydrophobic residues of
the sequence but rather they caused a neutralization of its net
positive charge. Thus, the binding of this sequence to calmodulin and
to the other Ca
-dependent proteins is not promoted
only by hydrophobic interactions as earlier proposed
(11, 12) , but it certainly also requires ionic
interactions. This proposal is supported by the EDC-catalyzed
cross-linking between calmodulin and the 145-163 sequence, we
described in this work, and between S100 protein and the CH22 fragment
recently reported
(35) . Although the NH
-terminal
segment Ala
-Phe
is still well conserved,
the eventual impact of the COOH-terminal amino acid replacements on the
actin recognition by the overall sequence Ala
-Val
is unknown. However, the recently identified co-localization of
the acidic calponin and actin in neuronal cells
(39)
(
)
and in various other non-muscle
tissues
(39) would argue for a normal in vivo complexation between the two proteins.
and/or Thr
which are thought to
represent the major sites of the in vitro phosphorylation of
calponin
(12, 13) . The phosphorylation event not only
inhibits the binding of calponin to actin
(4) but also it
significantly lowers its affinity for calmodulin
(40) and for
tropomyosin
(13) without suppressing the interaction of
calponin with the two latter proteins
(13) . These effects
obviously reflect the phosphorylation-mediated disruption of the
functioning of the actin site as well as CaT/CaM binding site 2 and TM
binding site 2 within the vicinal 145-163 sequence. In contrast,
the phosphorylation had no apparent influence on the activity of
CaT/CaM and TM binding sites 1 which remain operant in phosphorylated
calponin, thus explaining the reported binding of the latter protein to
immobilized calmodulin or tropomyosin
(4) . Conversely, the
binding of calponin to either F-actin, calmodulin, or tropomyosin was
shown to inhibit the phosphorylation reaction
(13, 33, 41) . The latter effect, which embodies
the three interactive functions of the 145-163 sequence, is in
agreement with our previous findings illustrating the ability of
F-actin to protect the peptide bound at Tyr
-Gly
against proteolysis
(18) . Owing to its various
interaction properties which are sensitive to calponin phosphorylation
at least in vitro, we anticipate that the 145-163 region
may represent an important regulatory domain of the protein. It lies
just in between the 13-kDa NH
- and 13-kDa COOH-terminal
fragments that we have earlier proposed to constitute two different
structural domains of calponin
(18) . Such an interdomain
location of the actin binding site was also observed in some other
actin-associated proteins like villin
(38) . It could confer to
the 145-163 sequence specific conformational features relevant to
the expression of its multiple functions.
Table:
Interaction of the synthetic peptides with
immobilized calmodulin or tropomyosin
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.