(Received for publication, July 10, 1995; and in revised form, November 16, 1995)
From the
A series of C-terminal deletion mutants of chicken gizzard smooth muscle caldesmon (CaD) were made using a polymerase chain reaction cloning strategy and a baculovirus expression system, and the precise locations of the functional domains of CaD involved in the regulation of actomyosin ATPase and the binding of actin, tropomyosin, and calmodulin were analyzed. Our results reveal a high affinity calmodulin-binding domain that consists of at least three calmodulin-binding determinants localized in residues 690-717, 658-689, and 628-657. The residues between positions 718 and 756 and positions 598 and 627 have no detectable calmodulin-binding site. A high affinity tropomyosin-binding domain is located between residues 718 and 756. The 159 residues at the C terminus of CaD contain multiple actin-binding determinants; the major ones are localized in the regions between residues 718 and 756 and residues 690 and 717. The amino acid residues between positions 718 and 756 contain the major determinant involved in the inhibition of the actin activation of smooth muscle myosin ATPase since CaD-(1-717) caused only 30% of the inhibition produced by the full-length CaD. Further deletion between residues 690 and 717 (CaD-(1-689)) revealed a low level (10% of that seen for full-length CaD) of inhibition of the actomyosin ATPase. These data clearly demonstrate that the region of the last 66 amino acid residues at the CaD C terminus contains two or more major actin-binding motifs, one tropomyosin-binding domain, one high affinity calmodulin-binding determinant, and the domain that is responsible for the inhibition of the actin-activated ATPase of myosin.
The thin filament-associated protein caldesmon (CaD) ()binds to actin, calmodulin, and tropomyosin, and the
binding of this protein to actin or tropomyosin-actin inhibits the
actin-activated Mg-ATPase activity of phosphorylated smooth muscle
myosin(1, 2, 3, 4, 5) .
This inhibition is released by the binding of calmodulin to caldesmon
in the presence of
Ca
(1, 2, 3, 6, 7) .
Furthermore, tropomyosin amplifies the caldesmon-induced inhibition of
actin-activated myosin ATPase activity (1, 3, 6) . CaD is thought to be a protein
involved in the thin filament-mediated regulation that complements the
myosin-mediated regulation of smooth muscle myosin via myosin light
chain phosphorylation and dephosphorylation ((8, 9, 10) ; for review, see (11) ).
Structurally, CaD is a long asymmetric monomeric (80 2 nm)
molecule that is localized along the actin filament, covering
7-14 actin monomers(12, 13, 14) . While
the CaD C terminus binds to actin, the N-terminal region of CaD binds
to the S-2 regions of the myosin(15, 16) . An
understanding of the structural and functional domains on the CaD
molecule, responsible for its interaction with myosin, actin,
calmodulin, and tropomyosin, is essential to elucidate the mechanism by
which CaD down-regulates actomyosin ATPase activity. Previous studies (17, 18, 19) have demonstrated an actin- and
a calmodulin-binding region on the C-terminal proteolytic fragment of
38 kDa (as determined by SDS-PAGE). The 38-kDa fragment possesses
the ability to inhibit actomyosin ATPase activity, but it fails to
cross-link myosin to actin (19, 20) . Using a
bacterial expression system, Redwood and Marston (21) made
several CaD fragments containing the sequences for actin- and
calmodulin-binding sites and for the region responsible for the
inhibition of the actin-activated ATPase activity of myosin. Since CaD
fragments, either derived from proteolysis or produced by a bacterial
expression system, lack a major portion of the molecule, they do not
produce the structural association between actin and caldesmon that is
likely to be associated with the thin filament-mediated regulation in
smooth muscle.
Recombinant CaD produced using the baculovirus
expression system is functionally and structurally similar to the
native molecule(22) . In this study, we utilize caldesmon
mutants made in a baculovirus expression system to characterize the
actin-, tropomyosin-, and calmodulin-binding domains and the ATPase
inhibitory domain on the CaD C terminus. Deletion of specific sequences
from the C-terminal region, without affecting the major portion of the
molecule responsible for the structural association with the actin
filament, enabled us to delineate the calmodulin-, actin-, and
tropomyosin-binding domains and to study the effect of these domains on
the actin-activated ATPase activity of smooth muscle myosin. We
demonstrate that the sequence that is present in the proteolytic CaD
fragment of 7.3 kDa (residues between Leu and
Phe
), thought to contain the region responsible for the
inhibition of the actin-activated ATPase of myosin S-1(12) , is
not important for the inhibition of the actin activation of smooth
muscle myosin ATPase in either the presence or absence of tropomyosin.
The region between residues 690 and 756 contains the high affinity
actin-binding site(s) and the domain that is important for the
inhibition of actomyosin ATPase activity. We also show that deletion of
a sequence (residues 718-725) previously reported to be important
for calmodulin binding to CaD, based on experiments using a bacterially
expressed CaD fragment(23, 24) , has no effect on
calmodulin binding.
Figure 1: Schematic representation of CaD C-terminal mutant proteins. The CaD mutant proteins that have deletions at the C terminus of CaD were generated using the baculovirus expression system. The numbers indicate the amino acid residue numbers. The hatched areas represent the sequence homology between CaD and troponin T characterized previously(35, 37) . The two cysteine residues on CaD molecule are presented as SH, and the central repetitive region consisting of a 13-amino acid sequence repeated eight times is indicated by the open bars(37) .
Figure 2:
SDS-polyacrylamide gels of unpurified or
purified mutants of CaD. A, Sf9 cell cultures infected with
the recombinant baculovirus vectors containing inserts of the sequence
for CaD mutants were solubilized with high salt lysis buffer (22) and centrifuged. The resultant supernatants were subjected
to SDS-PAGE followed by Coomassie Blue staining. Lane 1,
molecular mass standards (myosin, 200 kDa; -galactosidase, 116.3
kDa; phosphorylase b, 92.5 kDa; bovine serum albumin, 66.2
kDa; ovalbumin, 45 kDa; and carbonic anhydrase, 31 kDa); lane
2, purified CaD from chicken gizzard; lane 3, total
proteins in lysates of Sf9 cells infected with the wild-type
baculovirus; lanes 4-9, total proteins from Sf9 cells (5
10
) infected with CaD-(1-756),
CaD-(1-717), CaD-(1-689), CaD-(1-657),
CaD-(1-627), and CaD-(1-597) recombinant baculoviruses,
respectively. B. the CaD mutant proteins were purified from
heat-stable fractions from the cell extract by one-step ion-exchange
chromatography (DEAE-Sephacel column), and the amount, equivalent to
the yield from 0.5 ml of cell culture (5
10
cells),
of each of the purified CaD mutant proteins was analyzed by 4-12%
gradient SDS-PAGE followed by Coomassie Blue staining. Lanes 1 and 8, molecular mass standards (see A); lanes 2-7, CaD-(1-756), CaD-(1-717),
CaD-(1-689), CaD-(1-657), CaD-(1-627), and
CaD-(1-597), respectively. The second minor band in lane 1 in A and B is a proteolytic fragment of
myosin.
To obtain pure CaD mutants, Sf9 cells
(1-5 10
) were cultured in 1-5-liter
spinner flasks. Cells were infected with each of the recombinant
baculoviruses, and 36 h after infection, the cells were harvested,
homogenized, and heat-treated. The heat-stable fraction was collected
by centrifugation (18,000
g). The ammonium sulfate
fraction (25-50%) of the heat-stable extract was subjected to
further purification as described(19, 22) . All the
mutant proteins were purified to near-homogeneity (>95% purity) by
one-step ion-exchange chromatography using a DEAE-Sephacel
column(22) . The purified mutant proteins differ in molecular
size as shown by SDS-PAGE (Fig. 2B). As reported
previously for native caldesmon, the molecular sizes of full-length
CaD, CaD-(1-717), CaD-(1-689), CaD-(1-657),
CaD-(1-627), and CaD-(1-597) estimated by SDS-PAGE are
larger than their true molecular masses calculated based on the amino
acid sequence of CaD(35) .
Figure 3:
Binding of CaD mutants to smooth muscle
myosin. Smooth muscle myosin from chicken gizzard (2 µM)
was mixed with various concentrations of CaD-(1-756) (),
CaD-(1-717) (
), CaD-(1-689) (
),
CaD-(1-657) (
), CaD-(1-627) (
), and
CaD-(1-597) (
) in 45 mM KCl, 5 mM MgCl
, 0.1 mM CaCl
, 2 mM DTT, 2 mM ATP, and 10 mM imidazole HCl (pH 7.0)
for 10 min at room temperature, and the mixtures were cosedimented in
an Airfuge as described previously(28) . The proteins in the
supernatants and pellets were analyzed by SDS-PAGE and quantitated by
scanning densitometry.
Figure 4: Cosedimentation of CaD mutants with calmodulin-coated agarose. Fifty µl of calmodulin-coated agarose in equilibration buffer (see ``Results'') was incubated with equimolar amounts (4.5 µM) of each of the CaD mutants. The unbound and bound CaD mutants were processed for SDS-PAGE as described under ``Results'' and electrophoresed on gradient gels (4-15%). A, lane 1: molecular mass standards (see Fig. 2legend); lanes 2, 4, 6, and 8: CaD-(1-756), CaD-(1-717), CaD-(1-689), and CaD-(1-657) from washing, respectively; lanes 3, 5, 7, and 9: CaD-(1-756), CaD-(1-717), CaD-(1-689), and CaD-(1-657) from elution, respectively. B, lane 1: molecular mass standards (see Fig. 2legend); lanes 2, 4, 6, and 8: CaD-(1-756), CaD-(1-627), CaD-(1-597), and BSA from washing, respectively; lanes 3, 5, 7, and 9: CaD-(1-756), CaD-(1-627), CaD-(1-597), and BSA from elution, respectively. C, lanes 1 and 8: molecular mass standards (see Fig. 2legend); lanes 2, 4, and 6: CaD-(1-756), BSA, and smooth muscle tropomyosin from washing, respectively; lanes 3, 5, and 7: CaD-(1-756), BSA, and smooth muscle tropomyosin from elution, respectively. D, histogram of the relative levels of bound (hatched bars) and unbound (open bars) proteins from the binding studies determined by scanning densitometry. The upper lighter bands in BSA are contaminant proteins.
The binding
affinities of the CaD mutants for calmodulin were determined using
[C]iodoacetamide-labeled full-length CaD and CaD
mutants. As shown in Fig. 5, the specific binding of full-length
CaD to calmodulin increased upon raising the concentrations of CaD, and
the binding was saturated at
16 µM CaD. The
calmodulin binding of CaD-(1-717) was slightly lower than that of
full-length CaD, with concentrations in the range of 0-10
µM; however, the calmodulin binding of both full-length
CaD and CaD-(1-717) leveled off at a stoichiometry of
1 mol
of CaD/mol of calmodulin. Compared with that of full-length CaD,
deletion of the residues between positions 690 and 756
(CaD-(1-689)), 658 and 756 (CaD-(1-657)), and 628 and 756
(CaD-(1-627)) resulted in significant decreases in calmodulin
binding (43, 76, and 93%, respectively). The calmodulin binding of
CaD-(1-627) and CaD-(1-597) was close to that of the BSA
and tropomyosin used as negative controls. Scatchard analysis of CaD
binding to calmodulin-agarose was performed as
described(25, 36) . The apparent dissociation constant
for full-length CaD was 0.98 ± 0.061
10
M, similar to that of native CaD (K
= 0.87 ± 0.46
10
M). The calmodulin binding affinities of the mutants
were as follows: CaD-(1-717), K
=
1.21 ± 0.067
10
M;
CaD-(1-689), K
= 2.42 ± 0.058
10
M; and CaD-(1-657); K
= 3.47 ± 0.075
10
M.
Figure 5:
Binding of 14C-labeled
full-length CaD and CaD mutants to calmodulin. The binding of
calmodulin to full-length CaD and CaD mutants was determined at varying
concentrations of
C-labeled proteins. The moles of CaD
bound per mole of calmodulin are plotted as a function of CaD
concentrations. Conditions of assays and symbols are the same as
described for Fig. 3.
Figure 6:
Effect of CaD mutants on fluorescence of
pyrene-labeled tropomyosin. A constant concentration (0.25
µM) of pyrene-labeled tropomyosin was mixed with
0-1.5 µM full-length CaD or CaD mutants in 10 mM KCl, 10 mM MOPS (pH 7.0), and 2 mM DTT at 25
°C. The excitation wavelength was at 340 nm, and the emission
wavelength was at 485 nm. , CaD-(1-756);
,
CaD-(1-717);
, CaD-(1-689);
,
CaD-(1-657);
,
CaD-(1-627).
Figure 7:
Binding of CaD mutants to actin or
tropomyosin-actin. The binding of full-length CaD and CaD mutants to
smooth muscle actin or tropomyosin-actin was measured by
cosedimentation assays at 50 mM ionic strength (adjusted with
KCl) in 5 mM MgCl, 0.1 mM CaCl
, 2 mM ATP, 10 mM imidazole (pH
7.0), and 1 mM DTT at 25 °C. Actin concentration remained
constant at 25 µM, and tropomyosin was mixed with actin to
obtain a 1:4 molar ratio. A, binding of CaD mutants to actin; B, binding of CaD mutants to tropomyosin-actin. Symbols are as
described for Fig. 3.
In a parallel
experiment, we examined the binding of the CaD mutants to the
reconstituted tropomyosin-actin filament. As seen in Fig. 7(A and B), the presence of tropomyosin
increased the binding affinities of full-length CaD, CaD-(1-717),
and CaD-(1-689) for actin. In the presence of tropomyosin, the
apparent K value of actin binding for full-length
CaD (0.28 ± 0.015
10
M) was
2-fold lower than the K
value measured in the
absence of tropomyosin. Deletion of residues 718-756 caused a
significant increase in the K
value to
0.62
µM (0.62 ± 0.04
10
µM), which was 2.2-fold greater than the K
value obtained when full-length CaD was used.
The apparent K
values were 0.93 ± 0.047
10
M for CaD-(1-689), 2.08
± 0.065
10
M for
CaD-(1-657) and CaD-(1-627), and 2.45 ± 0.085
10
M for CaD-(1-597).
The
effect of Ca-calmodulin on the reversal of actin
binding was also determined. These results are shown in Fig. 8(A and B). The actin binding of all CaD
mutants decreased upon raising the concentration of calmodulin at a
constant 1:7 molar ratio of CaD to actin. At a 1:1 molar ratio of
calmodulin to actin (CaD/CaM ratio of 1:7), CaD released from actin was
76% for full-length CaD and CaD-(1-717), 60% for
CaD-(1-689), 56% for CaD-(1-657), and 20% for both
CaD-(1-627) and CaD-(1-597). The release of all CaD mutants
from actin by Ca
-calmodulin was less in the presence
of tropomyosin than in its absence (Fig. 8B). These
data suggest that tropomyosin increases the affinity of CaD for actin
due to an interaction between CaD and tropomyosin.
Figure 8:
Effect of Ca-calmodulin
on binding of CaD mutants to actin or tropomyosin-actin. Conditions are
the same as described for Fig. 6except that the concentrations
of CaD and actin were kept constant (0.15 mol of CaD/mol of actin). A, reversal of binding of CaD mutants to actin; B,
reversal of binding of CaD mutants to tropomyosin-actin. Symbols are as
described for Fig. 3. Note that the release of mutants
CaD-(1-597), CaD-(1-627), and CaD-(1-657) is not
remarkable since the binding of these mutants to actin is also very low
(see Fig. 7, A and B).
Figure 9:
Inhibition of actin-activated ATP
hydrolysis by smooth muscle myosin. The actin-activated and
tropomyosin-actin-activated myosin ATPase activities were measured at
25 °C in buffer at 50 mM ionic strength (adjusted with
KCl) containing 5 mM MgCl, 0.1 mM CaCl
, 2 mM ATP, 10 mM imidazole (pH
7.0), and 1 mM DTT. Smooth muscle actin and myosin were kept
constant at 25 and 1 µM, respectively, and tropomyosin was
mixed with actin at a 1:4 molar ratio. A, effect of CaD
mutants (CaD-(1-717)) on actin-activated ATPase activity; B, effect of CaD mutants on tropomyosin-actin-activated ATPase
activity. Symbols are as described for Fig. 3.
To determine whether the
inhibition of ATPase described above correlated with the binding of CaD
to actin either in the presence or absence of smooth muscle
tropomyosin, the binding assays and ATPase assays were carried out
simultaneously. As shown in Fig. 10(A and B),
the inhibition caused by full-length CaD and CaD-(1-717) was
40 and 15%, respectively, when
0.06 mol of CaD was bound per
mol of actin. However, in the presence of tropomyosin under the same
condition (0.06 mol of CaD bound per mol of actin), the inhibition
shown by full-length CaD and CaD-(1-717) was
52 and 25%,
respectively. These data suggest that tropomyosin enhances the
inhibition of the actin-activated myosin ATPase activity without
additional binding of CaD to actin.
Figure 10:
Relationship between CaD bound to actin
and inhibition of actin- or tropomyosin-actin-activated myosin ATPase
activity. Actin-activated ATP hydrolysis of phosphorylated smooth
muscle myosin was determined in the absence and presence of
tropomyosin. The binding of purified full-length CaD () and the
CaD mutant CaD-(1-717) (
) to actin (A) and
tropomyosin-actin (B) was determined in parallel experiments.
Conditions of the assays are as described for Fig. 9. A, relationship between the inhibition of actin-activated
ATPase activity and the actin binding of full-length CaD and the CaD
mutant CaD-(1-717) in the absence of tropomyosin; B,
relationship between the inhibition of actin-activated ATPase activity
and the actin binding of full-length CaD and the CaD mutant
CaD-(1-717) in the presence of
tropomyosin.
Our data utilizing these truncated CaD proteins reveal some important differences from the data obtained from studies using bacterially expressed or proteolytic CaD fragments, presumably due to the lack of structural association present in protein-protein interaction. The major myosin-binding site is located in the N terminus of CaD(15, 39) , and a recent study shows that the residues between positions 235 and 531 of rat nonmuscle CaD coprecipitate with smooth muscle myosin in an in vitro binding assay(40) . This finding is further supported by Marston and co-workers(41) , who showed that a bacterially produced fragment composed of the 288 residues at the C-terminal region of human nonmuscle CaD bound to myosin. But the ability of this fragment to bind to myosin was not compared with that of CaD containing the central helical region and parts of the C terminus. In the present study, a slight decrease (6, 11, and 13% for CaD-(1-717), CaD-(1-627), and CaD-(1-597), respectively) in the binding of caldesmon mutants to myosin was observed when compared with the full-length molecule. This is probably caused by the conformational change in the rest of the CaD molecule. Maximal binding of CaD to myosin seems to require the C-terminal region of CaD to be intact.
Studies using chymotryptic peptides of CaD have demonstrated that
the C-terminal region of CaD contains a calmodulin-binding site,
presumably in the region between residues 629 and 666(37) .
Zhan et al.(42) showed that a synthetic peptide with
an amino acid sequence homologous to CaD between residues 658 and 666
bound to calmodulin. A recent report showed that a synthetic peptide
spanning the residues between positions 675 and 695 bound to
calmodulin-Sepharose and was eluted with EGTA(43) . These data
indicate the possibility that another calmodulin-binding site exists in
the C-terminal end of CaD. Data from the present study reveal that the
C-terminal high affinity calmodulin-binding site of chicken gizzard
smooth muscle CaD is located in the last 129 amino acids, probably
involving two or more discontinuous epitopes. The residues between
positions 690 and 717 and positions 658 and 689 play a major role in
maintaining high affinity binding to calmodulin since deletion of
either of these regions results in a significant decrease in calmodulin
binding when compared with that of full-length CaD. These data further
confirmed the results from analyses of synthetic
peptides(42, 43) , which determined that the last 99
amino acid residues contained two calmodulin-binding determinants. It
has been proposed that the core regions of the two calmodulin-binding
determinants more likely involve residues 658-666
(calmodulin-binding motif 1) (42) and residues 675-695
(calmodulin-binding motif 2)(43) , respectively. Based on the
information from the data in the present study and on comparison with
data from published reports(43) , the core region of
calmodulin-binding motif 2 can be further defined to a 5-amino acid
stretch from Asn to Lys
(Fig. 11).
Our results also show that the residues between positions 628 and 657,
part of the sequence included in the region indicated by Wang et
al.(37) as the partial sequence for calmodulin binding on
the CaD molecule, do not contain a region responsible for the high
affinity binding of CaD to calmodulin. However, CaD-(1-657) binds
weakly to calmodulin, but CaD-(1-627) does not ( Fig. 4and Fig. 5), indicating that the region between residues 628 and 657
contains a sequence responsible for weak calmodulin binding. In
addition, our data rule out a calmodulin-binding site in the region
between residues 718 and 756, a region suggested by Marston and
co-workers (23, 24) to be important for calmodulin
binding. The difference in the conformation between the truncated
proteins containing the N-terminal and central helical regions used in
the present study and the caldesmon fragments used in previous studies
may account for this discrepancy. Calmodulin binding is similar for
truncated CaD-(1-717) and full-length CaD (Fig. 5).
However, in the presence of CaM, the dissociation of full-length CaD
and CaD-(1-717) from actin is the same, despite the weak binding
of the latter to actin. This suggests that calmodulin dissociates the
bound CaD from actin by inducing a similar conformational change in the
regions between residues 690 and 717 and residues 718 and 756.
Figure 11: Summary of domain structure at C terminus of CaD. The region between residues 508 and 565 is homologous to the tropomyosin-binding site of troponin T and is considered to be the tropomyosin-binding site on the CaD molecule(37) . High affinity calmodulin (CaM)-binding site 1 (MWEKGNVFS) is derived from Wang and co-workers(42) , and high affinity calmodulin-binding site 2 is deduced from our present data and the published data of Mezgueldi et al.(43) . The tropomyosin-binding domain was localized in the region between residues 718 and 727 ((47) ).
It is
well known that tropomyosin, a coiled-coil thin filament-associated
protein, can directly bind to CaD with a stoichiometry of 1-2 mol
of tropomyosin/mol of CaD(13, 14, 44) .
Electron microscopic observations of reconstituted thin filament
revealed that CaD fixes tropomyosin in a particular position on actin
filaments(45) . However, there is very little information on
the tropomyosin-binding sites on CaD. Based upon sequence alignment
analysis with the tropomyosin-binding site on troponin T, the
tropomyosin-binding site on chicken gizzard smooth muscle CaD is
proposed to lie between residues 508 and 565(35, 37) .
Our data with truncated CaD show that the deletion of 39 residues at
the C-terminal end of CaD remarkably reduces the binding of CaD to
tropomyosin (Fig. 6), in contrast to the report by Redwood and
Marston (21) showing that there is no tropomyosin-binding site
between residues 658 and 756. Further removal of up to 120 residues
from Asn to Gly
(as in CaD-(1-597),
CaD-(1-627), CaD-(1-657), and CaD-(1-689)) caused
only a slight decrease in the binding to tropomyosin. These results
suggest strongly that CaD contains two or more tropomyosin-binding
sites; the major tropomyosin-binding site apparently resides in the
C-terminal 39 amino acids. It appears that the region between amino
acids 627 and 717 of CaD is not involved in the binding of tropomyosin
since only a small difference is observed between CaD-(1-627) and
CaD-(1-717) in tropomyosin binding.
This study is in agreement with our previous finding that tropomyosin can enhance the binding of CaD to actin via a triggering of conformational changes on these thin filament-associated proteins(33) . The binding of full-length CaD and CaD-(1-717) or CaD-(1-689) to actin was higher in the presence of tropomyosin than in its absence (Fig. 7A). It should be noted that the actin binding of CaD-(1-657), CaD-(1-627), and CaD-(1-597) can be slightly enhanced by tropomyosin (Fig. 7B), indicating that a tropomyosin-binding site present in the region between residues 1 and 597, perhaps in the region between residues 230 and 419 of CaD shown to bind to tropomyosin(21) , is also responsible for the tropomyosin-enhanced binding of CaD to actin.
The major
actin-binding site(s) on the CaD molecule has been traced to the
C-terminal region of CaD, perhaps between residues 659 and 665 (12) or involving two independent binding motifs that are
separated by residues 629-666(37) . Our cosedimentation
binding assays reveal that there are a number of actin-binding motifs
present within the last 159 amino acids at the C terminus of chicken
gizzard CaD. CaD-(1-717), which lacks 39 amino acids, shows
diminished binding to actin, suggesting that the residues between
positions 718 and 756 contributed to the high affinity actin-binding
site as postulated by Wang et al.(37) . However,
another crucial actin-binding determinant has been found to lie between
residues 690 and 717 (Fig. 7A). It is possible that
these sequences are components of one binding site with more than one
determinant. The observation that CaD-(1-657) weakly bound to
actin rules out the possibility that the region between residues 597
and 629 (37) is attributable to the high affinity actin binding
of CaD. As described in our previous report(22) ,
CaD-(1-597), which lacks 156 residues at the C terminus of CaD,
weakly binds to actin. However, the apparent K value for CaD-(1-597) was 5-fold higher than that for
full-length CaD, indicating that the region between amino acids 1 and
597 contains an additional low affinity actin-binding site.
Interestingly, an actin-binding site in residues 483-508 has been
proposed by Wang et al.(37) , and a recent electron
microscopic image (46) also provides direct evidence that both
ends of CaD interact with actin filaments.
It has been well established that CaD inhibits the actin-activated or tropomyosin-actin-activated ATPase activity of both myosin I and myosin II(1, 2, 3, 4, 5, 38) . Several lines of evidence show that the inhibitory region is located in the last 99 amino acids at the C terminus of CaD (19, 21) . Our present study reveals that there are at least two ATPase inhibitory determinants in the C-terminal region of chicken gizzard smooth muscle CaD. These determinants are between residues 718 and 756 and residues 690 and 717 (Fig. 9, A and B). The residues between positions 658 and 689, which contain most of the amino acid sequence of the 7.3-kDa chymotryptic fragment(12) , cause only a very weak ATPase inhibition (see inhibition by CaD-(1-689) and CaD-(1-657) in Fig. 9). In contrast, the domain between residues 718 and 756 contains the critical determinants for the ATPase inhibition since CaD-(1-717), lacking the residues between positions 718 and 756, displays 30% of the ATPase inhibition produced by full-length CaD (Fig. 9). As expected, both CaD-(1-627) and CaD-(1-597) did not elicit any effect on the actin-activated ATPase activity, consistent with previous reports(21, 22, 37) . These data indicate that the sequences between residues 690 and 717 and residues 718 and 756 contain the essential regulatory motifs for the actin-activated ATPase activity, and these regulatory motifs are functionally independent or separate. Our present results are in agreement with the observation of Wang et al.(37) that the last 39 amino acid residues of CaD are mainly responsible for the inhibition of the actin activation of myosin ATPase. These findings conflict with the observation of Chalovich et al.(12) that the residues between positions 597 and 665 encompass the major inhibitory sequences for actomyosin ATPase. The reason for this discrepancy is not clear; interestingly, this proteolytic fragment bound to actin at a 1:1 molar ratio, and the inhibition of actomyosin ATPase required a very high CaD fragment (7.3 kDa) to actin molar ratio, although the intact CaD molecule covers between 7 and 14 actin monomers.
Tropomyosin-dependent ATPase inhibition was also tested in our study. The ATPase inhibition caused by CaD-(1-717), but not by CaD-(1-689), was potentiated by tropomyosin, suggesting that the region between residues 690 and 756 is related to the tropomyosin-dependent ATPase inhibition. At the same molar ratio of CaD to actin, the percent inhibition in the presence of tropomyosin is higher than that in its absence (Fig. 10, A and B).
In conclusion, as shown in Fig. 11, the last 66 amino acid residues at the C terminus of CaD contain two or more major motifs for actin binding, a tropomyosin-binding determinant, and one high affinity calmodulin-binding determinant, of which the last 39 amino acid residues are involved in both actin binding and tropomyosin binding. The tropomyosin-binding motif in the region consisting of the last 39 amino acids can be further defined to a 10-amino acid stretch between residues 718-727 using a C-terminal deletion mutant that lacks residues between positions 728 and 756(47) . This region is also responsible for the major inhibition of the actin-activated ATPase activity of myosin. The 28-amino acid stretch (residues 690-717) encompasses both low affinity actin binding and high affinity calmodulin binding. These results suggest that the calmodulin-, actin-, and tropomyosin-binding motifs on the CaD molecule are overlapping or are closely juxtaposed with each other. The localization of these important motifs at the C terminus of CaD helps add to the basic understanding of the structural and functional domains on the CaD molecule involved in the regulation of actin-activated ATP hydrolysis by myosin.