(Received for publication, August 25, 1995; and in revised form, December 16, 1995)
From the
Tropomyosins are highly conserved, coiled-coil actin binding
proteins found in most eukaryotic cells. Striated and smooth muscle
-tropomyosins differ by the regions encoded by exons 2 and 9.
Unacetylated smooth tropomyosin expressed in Escherichia coli binds actin with high affinity, whereas unacetylated striated
tropomyosin requires troponin, found only in striated muscle, for
strong actin binding. The residues encoded by exon 9 cause these
differences (Cho, Y.-J., and Hitchcock-DeGregori, S. E.(1991) Proc.
Natl. Acad. Sci. U. S. A. 88, 10153-10157). We mapped the
functional domains encoded by the
-tropomyosin exon 9a (striated
muscle-specific) and 9d (constitutively expressed), by measuring actin
binding and regulation of the actomyosin MgATPase by tropomyosin exon 9
chimeras and truncation mutants expressed in E. coli. We have
shown that: 1) the carboxyl-terminal nine residues define the actin
affinity of unacetylated tropomyosin; 2) in the presence of
Ca
, the entire exon 9a is required for troponin to
promote fully high affinity actin binding; 3) the first 18 residues
encoded by exon 9a are critical for the interaction of troponin with
tropomyosin on the thin filament, even in the absence of
Ca
. The results give new insight into the structural
requirements of tropomyosin for thin filament assembly and regulatory
function.
Tropomyosin (TM) ()is an actin binding protein, first
discovered in skeletal muscle(1) , and now known to be present
in virtually all eukaryotic cells. Tropomyosins are a family of highly
conserved, coiled-coil proteins in which diversity is primarily
accomplished through the use of alternative promoters and alternative
RNA splicing of the transcripts of a small number of genes (three to
four in vertebrates; reviewed in (2) ).
Tropomyosins are
expressed in developmental and tissue-specific patterns. Certain
isoforms are found in more than one cell type and tissue, although they
may have different localizations within cells (e.g.(3) ). Other TM isoforms are specific, notably those
expressed in striated and smooth muscles (striated and , smooth)
and the specific brain
-TM isoforms ( (4) and (5) ; reviewed in (2) ). These isoforms contain
uniquely-expressed exons that presumably encode domains of the protein
involved in isoform-specific function.
The functions of TM are best understood from the extensive in vitro biochemical and biophysical analysis since the protein's discovery. Two fundamental functions that define TMs are cooperative binding to F-actin (6) and cooperative inhibition or activation of the actomyosin MgATPase(7) . Tropomyosin isoforms differ in these assays (reviewed in (8) ), and their activities are influenced by other cytoskeletal proteins that interact directly or indirectly with TM (caldesmon, troponin (Tn), and myosin).
The cellular
function of TM is best understood in striated muscles where, through
interaction with Tn, it regulates contraction in a
Ca-dependent manner (reviewed in (9, 10, 11) ). Biochemical and structural
studies by numerous investigators have led to the following model of
the thin filament. Tropomyosin molecules are aligned end-to-end
(NH
terminus to COOH terminus) in the grooves of the
helical actin filament(12, 13) . Troponin is bound to
TM along the COOH-terminal third of the molecule (14, 15) . Troponin I, TnC, and the COOH terminus of
TnT are positioned near Cys-190 of TM. The elongated NH
terminus of TnT extends toward the COOH terminus of TM to the
beginning of the next TM along the actin filament. Troponin greatly
increases the affinity of TM for actin in the presence of
Ca
, with a further increase upon removal of
Ca
(16) . Based on binding studies with TM and
Tn peptides, Mak and Smillie (17) proposed that the Tn-TM
interaction in the region of Cys-190 is Ca
-sensitive
(stronger in the absence of Ca
), whereas that at the
COOH terminus and the overlap region is
Ca
-independent (strong both in the presence and
absence of Ca
). Substantial evidence has since
supported this model.
The Ca-independent TnT
binding site on TM includes the COOH-terminal 27 amino acids encoded by
the striated muscle-specific exon 9a, implying a specific role in
Tn-dependent regulatory function. Exon 9a is uniquely expressed in the
striated isoform whereas the COOH-terminal coding exon of most other
-TM isoforms, is exon 9d (18; reviewed in (2) ). The
functional differences between these 284 residue
-TMs are
primarily attributable to the exon 9-encoded COOH
terminus(19) . Unacetylated striated
-TM expressed in Escherichia coli binds poorly to F-actin because the
NH
-terminal Met is unacetylated (20, 21, 22) whereas TMs encoded by cDNAs
containing exon 9d (smooth and TM2, a non-muscle isoform) bind
well(19) . In the presence of Ca
, Tn greatly
increases the affinity of the unacetylated striated TM for actin but
has little effect on unacetylated smooth TM or TM2(19) .
Similar differences have been reported between unacetylated smooth and
striated
-TMs(23, 24) .
In the present work we
have defined the functional domains encoded by -TM exons 9a and 9d
(two of the four COOH-terminal
-TM exons) by making a series of TM
chimeras and truncation mutants. Unacetylated TMs expressed in E.
coli offer a powerful system for looking at gain or loss of TM
functions: actin affinity and Ca
-independent
interaction with Tn on the actin filament. We have shown that: 1) the
actin affinity of unacetylated TM is defined by the COOH-terminal nine
amino acids; 2) the entire striated-specific exon 9a is required for Tn
to promote fully high affinity binding of TM to actin in the presence
of Ca
; and 3) the first 18 residues encoded by exon
9a are critical for the interaction of Tn with TM on the thin filament,
even in the absence of Ca
. The results, together with
previous work from our laboratory and others, indicate that the
NH
- and COOH-terminal ends of TM are primary functional
determinants. The results are discussed in terms of the structural
requirements of TM for regulatory function with Tn. Portions of this
work have been reported in a preliminary form (25) .
Rat striated (pUC18/TM9a) and rat smooth (pBR322/SMTM)
muscle -TM cDNA clones were the gift of B.
Nadal-Ginard(4) . The coding regions differ only in exons 2 and
9 corresponding to codons 39-80 and codons 258-284,
respectively, with codon number 1 defined as the ATG immediately
following the NcoI cleavage site.
To facilitate a subsequent cloning step, both striated and smooth cDNAs were subcloned into pUC119, destroying the vector's SmaI site (Fig. 1A). In order to accomplish this, TM9a cDNA was digested with EcoRI. The 1212-base pair fragment was isolated from an agarose gel, after which the 5` overhang was filled in using Klenow. This blunt fragment was ligated to the SmaI site of pUC119 resulting in the vector, pUC119/TM9a. The smooth tropomyosin cDNA was digested with PstI. The 1078-base pair fragment was isolated from an agarose gel, after which the 3` overhangs were filled in using T4 DNA polymerase. This blunt fragment was ligated to the SmaI site of pUC119 resulting in the vector, pUC119/SMTM. The NcoI-SmaI fragment from pUC119/TM9a containing TM codons 1-237 was ligated to the NcoI-SmaI fragment from pUC119/SMTM containing TM codons 238-284. The resulting chimera is pUC119/TM9d.
Figure 1: Construction of tropomyosin exon 9 variant plasmids (see ``Materials and Methods'' for details). A, striated (TM9a) and smooth (SMTM) cDNAs were subcloned into pUC119 in order facilitate future cloning steps. The NcoI-SmaI fragment encoding TM9a codons 1-237 was ligated to the NcoI-SmaI fragment encoding SMTM codons 238-284 resulting in TM9d. B, to generate TMs with chimeric ninth exons (TM9a/9d and TM9d/9a), the EcoRI-AvaII fragments of TM9a and TM9d containing TM codons 1-275 and the AvaII-BamHI fragments of TM9a and TM9d containing codons 276-284 were exchanged then ligated with pUC119. C, tropomyosin truncation mutants with the 3` nine codons deleted were constructed by filling in the 5` overhangs of the EcoRI-AvaII fragment (B) with Klenow then ligating to a BamHI linker. After digesting with NcoI and BamHI, this fragment was ligated to the expression vector, pET11d(35) .
To construct TMs which contain chimeric ninth exons (TM9a/9d, TM9d/9a), pUC119/TM9a and pUC119/TM9d were digested with AvaII (Fig. 1B). The fragments containing TM codons 1-275 were further digested with EcoRI, and the fragments containing TM codons 276-284 were further digested with BamHI. The fragments containing TM codons 1-275 and TM codons 276-284 of pUC119/TM9a and pUC119/TM9d were exchanged and then ligated with pUC119 which had been linearized with EcoRI and BamHI. The result is pUC119/TM9a/9d and pUC119/TM9d/9a.
Truncation mutants were constructed by deleting the cDNA coding for the carboxyl-terminal 9 residues of TM (Fig. 1C). This was accomplished by filling in the 5` overhang of the AvaII fragment isolated from pUC119/TM9a using Klenow and ligating it to a linker (TGATAAGGATCCTTATCA) containing termination codons (underlined) followed by a BamHI site (in italics). After digestion with NcoI and BamHI, the fragment was ligated to the NcoI and BamHI sites of the pET11d expression vector (27) resulting in pET11d/TM9a/. pUC119/TM9d was treated in the same manner resulting in pET11d/TM9d/.
The NcoI-BamHI fragments of pUC119/TM9a, pUC119/TM9d,
pUC119/TM9d/9a, and pUC119/TM9a/9d were ligated to the NcoI
and BamHI sites of the expression vector, pET11d. The cDNAs
for all six TM variants in pET11d were expressed in E. coli (BL21{DE3}pLysS for TM9a or BL21{DE3} for the
other TM variants)(27) . The TMs were purified from 2 liters of
cells (yield = 20-30 mg/liter) as described previously (20) except the (NH)
SO
fractionation was 35-70% saturation instead of
35-60%.
Actin was purified from acetone powder of pectoral
muscle of White Leghorn chickens according to (28) , except
actin was polymerized by addition of KCl and MgCl to a
final concentration of 20 and 0.7 mM, respectively, and
incubated at 37 °C for 10 min before cooling slowly to room
temperature.
Troponin was purified from pectoral muscle pulp of
White Leghorn chickens obtained from Dr. J. Fagan (Rutgers University,
NJ) using the procedure of Potter (29) except, alternate Triton
X-100 extractions included the following protease inhibitors: 2 mM E64, 1 mg/ml leupeptin; 2 mM 4-amidinophenylmethane-sulfonyl fluoride, 0.5 M benzamidine. Also, the (NH)
SO
crude Tn pellet was dialyzed against 20 mM Tris, pH 7.5,
0.1 mM CaCl
, and 0.5 mM dithiothreitol
(DTT) then purified using ion exchange (DE52) column chromatography
with a 0-0.6 M NaCl gradient. Chicken pectoral muscle
myosin was isolated using the method of Margossian and
Lowey(30) .
The isoform-specific differences in actin affinity and
interaction with Tn of recombinant, unacetylated 284 residue -TMs
can be primarily attributed to differences in residues 258-287,
encoded by exon 9(19) . To investigate the regions encoded by
exons 9a and 9d (18; reviewed in (2) ) that give rise to the
isoform-specific function, we made a series of chimeras and deletion
mutants, illustrated in Fig. 2A, in which the last nine
residues have been exchanged or deleted. The sequences of the variants
are identical except for the residues encoded by the two ninth exons,
shown in Fig. 2B. The deletion mutant TM9a/ is similar
to carboxypeptidase-digested TM in which the last 11 residues are
removed enzymatically. These six variants were expressed in E. coli as unacetylated TMs which offer the opportunity to study gain or
loss of TM-specific functions: high affinity actin binding, the ability
of Tn to increase the affinity of TM for actin, and Ca
regulation of the actomyosin ATPase with Tn.
Figure 2:
Tropomyosin exon 9 variants. A,
all variants are identical to striated rat -TM except for changes
in the ninth exon represented by boxes. Numbers indicate the amino acid position at the boundaries of the changes. B, comparison of rat
-TM exon 9a and 9d encoded
sequences. Identities are enclosed in boxes; the positions in
the heptapeptad repeat are shown in lower case
letters.
Figure 3:
Actin binding of tropomyosin exon 9
variants. Conditions: TM was cosedimented with actin (5
µM) at 25 °C in 150 mM NaCl, 10 mM Tris, pH 7.5, 2 mM MgCl, 0.5 mM DTT.
, TM9a/9d;
, TM9d;
, TM9a;
, TM9d/9a;
,
TM9d/;
, TM9a/. The TM/actin ratio determined using densitometry
(arbitrary units) was normalized using the n reported by
SigmaPlot using the Hill equation (see ``Materials and
Methods''). The data shown are from three experiments for TMs
which bind (TM9a/9d, TM9d) and from one to two experiments for TMs
which do not bind (TM9a, TM9d/9a, TM9d/,
TM9a/).
Fanning et al.(31) reported that unacetylated
striated -TM purified using a modified method (see
``Materials and Methods'') binds actin strongly in the
presence of 100 mM KCl. However, we found that unacetylated
striated
-TM purified by the Fanning method (31) bound
poorly to actin in 100 mM NaCl (K
< 1
10
M
) whereas the same
protein purified using the Hitchcock-DeGregori method (20) bound with measurable affinity (K
1
10
M
),
suggesting that the modified purification method adversely affects TM
function. Also, acetylated muscle TM repurified using the
Hitchcock-DeGregori method (20) bound actin with high affinity,
indicating that this method does not alter the TM itself. These results
confirm that the weaker actin affinity of striated
-TM purified
from E. coli is a consequence of the lack of
NH
-terminal acetylation and not the purification
method(20) .
Figure 4:
Actin binding of tropomyosin exon 9
variants with Tn in the presence (A) and absence (B)
of calcium. Conditions: TM and Tn (2 µM) were cosedimented
with actin (5 µM) at 25 °C in 150 mM NaCl, 10
mM Tris, pH 7.5, 2 mM MgCl, 0.5 mM DTT, 0.2 mM CaCl
, or 0.2 mM EGTA.
, TM9a/9d;
, TM9d;
, TM9a;
, TM9d/9a;
,
TM9d/;
, TM9a/. The TM/actin ratio determined using densitometry
(arbitrary units) was normalized using the n reported by
SigmaPlot using the Hill equation (see ``Materials and
Methods''). The data shown in A are from three
experiments for TMs which bind (TM9a/9d, TM9d, TM9a, TM9d/9a) and from
one to two experiments for TMs which do not bind (TM9d/, TM9a/). In B, data are from two experiments for TMs which bind (TM9d/9a,
TM9a/) and from one experiment for TMs which bind too tightly to
measure (TM9a/9d, TM9d, and TM9a) or do not bind
(TM9d/).
With Tn in the absence of
Ca, the affinity of all TM exon 9 variants was
greater, with the exception of TM9d/ (Fig. 4B, Table 2). The increase in affinity was in the order of 10-fold,
consistent with our previous measurements(19) , although
certain variants bound too tightly to measure accurately in the
conditions of these experiments. TM9a/, whose affinity was too weak to
measure in the presence Ca
, bound well in the absence
of Ca
, as previously reported for
carboxypeptidase-treated unacetylated TMs(42) .
Most
interesting, however, is that TM9d/ did not bind to actin, even in the
absence of Ca while TM9a/ did. Since the Cys-190
region, which includes the Ca
-sensitive binding site
of Tn on TM (17) is identical in all exon 9 variants studied,
we expected that Tn-TM interaction in the absence of Ca
would be the same in TM9a/ and TM9d/. Clearly, the binding of Tn
only to the Cys-190 region of TM is insufficient to assemble Tn-TM on
the actin filament, even in the absence of Ca
. The
results suggest that the first 18 residues of exon 9a are important for
the increased affinity of Tn-TM for actin in the absence as well as in
the presence of Ca
.
The binding constants for TM9a
and TM9d reported here are higher than those previously
published(19) . The present TMs have been expressed using a
different expression system with higher yields and better purity. We
have experienced similar measurements of increased affinity in other
recombinant TMs. ()
Figure 5:
Effect of tropomyosin exon 9 variants on
the actomyosin Mg ATPase in the presence of Tn
without Ca
. Conditions: 2.4 µM actin,
0.6 µM myosin, 0-0.6 µM TM, 1
µM Tn in 40 mM NaCl, 5 mM imidazole, pH
7.0, 0.5 mM MgCl
, 5 mM MgATP, 1 mM DTT, 0.2 mM EGTA for 15 min at 28 °C. Specific
activity is expressed as micromoles of P
/mg of myosin/min.
In the absence of TM, the mean specific activity of the actin-Tn-myosin
ATPase for all experiments pooled was 0.15 ± 0.01 µmol of
P
/mg of myosin/min (range = 0.13-0.17 µmol
of P
/mg of myosin/min; n = 26). Each curve
contains normalized data pooled from two to five experiments. The data
were normalized to the mean specific activity of the actin-Tn-myosin
ATPase in the absence of TM for each experiment. Only TM9a is shown
with Tn and 0.2 mM Ca
, since it is
representative of all the TM exon 9
variants.
In the present work we have defined the functional domains
encoded by exon 9a, a striated muscle-specific exon, and exon 9d, the
constitutively-expressed exon found in smooth and most non-muscle
-TMs. The COOH-terminal nine amino acids of TM9d allow
unacetylated TM to bind to actin with high affinity. The exon
9a-encoded COOH terminus, especially the first 18 residues, confers an
isoform-specific binding site for Tn which is essential for
Tn-dependent thin filament assembly of unacetylated TM. The
COOH-terminal domain is the most variable among TM isoforms; it is
encoded by alternative exons (four in
-TMs) and it is more
variable between genes and species than much of the rest of
TM(2, 44) .
We have shown that the last nine amino
acids are fully responsible for the differences in actin affinity
(without Tn) between unacetylated TM9a (striated TM; low affinity for
actin) and TM9d (TM2, a non-muscle isoform; high affinity for actin).
Comparison of the amino acid sequences of the last nine residues
encoded by exons 9a and 9d (Fig. 2B) shows that there
is only one identity (Leu-278) and three conservative changes. At this
point it is difficult to predict the involvement of specific residues
in the observed changes in actin affinity. Interestingly, Pittenger et al.(45) reported that striated -TM (exon 9a)
binds to actin with higher affinity than smooth
-TM (exon 9d)
although the amino acid sequences of the last nine residues encoded by
-TM exons 9a and 9d differ from the corresponding
-TM ninth
exons by only one to two conservative changes.
Our second major
conclusion is that the entire striated-specific exon 9a is required for
Tn to promote fully high affinity binding of TM to actin in the
presence of Ca. The significant increase in affinity
of the exon 9 chimeras, compared to TM9d, shows that both parts of exon
9a contribute, but that the effect is greatest with all 27 exon 9a
residues. If only the first 18 residues of exon 9a were necessary for
this strong (100-fold) promotion of binding, we would expect Tn in the
presence of Ca
to increase the actin affinity of
TM9a/9d by more than the observed 4-fold effect. Although TM9a and
TM9a/9d bind to TnT with similar affinity when measured using a solid
phase affinity assay (BIAcore, Pharmacia) (
)the ternary Tn
complex in the presence of Ca
only promotes the actin
affinity of TM9a. Therefore, the first 18 residues encoded by exon 9a
are sufficient for TnT binding, whereas both parts are necessary for
promotion of TM binding to the regulated thin filament in the presence
of Ca
.
Another possible interpretation of the
failure of Tn to greatly increase the actin affinity of TM9d or TM9a/9d
in the presence of Ca is that they bind well in the
absence of troponin and the effect of Tn is not additive. We do not
favor this interpretation as previous studies have shown that the
effect of Tn on striated muscle (acetylated)
-TM, which binds well
in the absence of Tn, is additive, greatly increasing the affinity in
the presence of Ca
(16, 37) .
Exon
9a encodes what has long been recognized as the
Ca-insensitive binding site of Tn for
TM(17) , and our results support this model. Our finding that
Tn in the presence of Ca
does not promote actin
binding of TM9a/ is consistent with previous analyses of
carboxypeptidase-treated TM from which the last 11 residues were
removed(42, 46) . Removal of the last 11 residues of
TM weakens the binding of the NH
-terminal region of TnT
(residues 1-158) to TM(17, 47, 48) ,
indicating the involvement of the entire COOH-terminal region of TM for
Tn binding in the presence of Ca
.
TM9d/ did not
bind measurably to actin with Tn in the absence of
Ca. We found this surprising since a reconstituted Tn
complex with residues 159-259 of TnT binds to a TM affinity
column (49) and promotes the actin binding of
carboxypeptidase-treated striated
-TM in the absence of
Ca
(48) . The complex of TnC, TnI, and
residues 159-259 of TnT is known to bind to TM in the region of
Cys-190, referred to as the Ca
-sensitive binding
site, and has not been considered to bind to the extreme COOH terminus
of TM(17, 48, 49, 50, 51) .
The different affinities of TM9a/ and TM9d/ (which have the same
sequence in the Cys-190 region) for regulated actin in the absence of
Ca
presumably reflect the differences in Tn affinity
for the COOH terminus.
The properties of TM9d/ are comparable to TM
with larger COOH-terminal deletions. A fusion striated -TM lacking
the last 39 residues does not bind to actin with Tn in the presence or
absence of Ca
(52) . Neither striated nor
smooth
-TM with the last 27 residues deleted bound well to
actin(45) . Striated
-TM with the last 31 residues deleted
does not bind to actin with Tn in the presence of Ca
,
although it retains limited ability to regulate the actomyosin S1
ATPase(23) . The loss of function of TM9d/ is as great as that
observed when the first nine residues are deleted from striated
-TM(53) . Troponin T extends across the overlap region and
binds to the NH
terminus of the next TM on the thin
filament(15, 54) . We suggest that the 27
COOH-terminal residues encoded by exon 9, the first 18 in particular,
together with the extreme NH
terminus of TM are critical
for striated TM-Tn assembly on the actin filament. In our analysis, it
is not possible to distinguish between altered binding of TM directly
to actin and altered end-to-end TM interactions on the thin filament.
All unacetylated TMs polymerize poorly, even with
Tn(19, 20) .
Comparing the sequences encoded by the first 18 codons of exons 9a and 9d (Fig. 2B), there are only three identities and four conservative changes; the striated form is slightly more acidic. In the absence of high resolution structural information, the sequences do not contribute to our understanding of the functional differences between the TM isoforms.
The ability of
the TM exon 9 variants to regulate the actomyosin ATPase with Tn was
insensitive to the observed differences in actin affinity. This is not
surprising since at the concentrations used in the assays the thin
filament would be fully saturated. Only TM9d/ was unable to regulate,
an expected result since it did not bind to the thin filament. A more
sensitive assay would be the cooperative activation of the thin
filament by myosin S1, the extent of which is TM isoform
specific(55, 56) . Unfortunately, unacetylated
284-residue TMs do not exhibit this
activation(19, 23) , and in the presence of Tn and
Ca, the activation of the acto-S1 ATPase is similar
with acetylated striated
-TM, unacetylated striated
-TM, and
unacetylated smooth
-TM(19, 53) . While we
anticipate the COOH-terminal differences will contribute significantly
to cooperative regulation by myosin S1, investigation of this TM
function will require acetylated TMs produced in insect Sf9
cells(21) , the subject of future experiments.
Our results
have given us insight into the structural requirements of TM for thin
filament assembly and regulatory function. We now understand the
functional significance of the striated-specific COOH terminus encoded
by exon 9a in terms of a long-standing model of regulation. The binding
of Tn in the Cys-190 region of TM is known to be stronger in the
absence than in the presence of Ca, but in both
conditions it is weaker than the binding of TnT to the COOH-terminal
region of TM(47, 57) . Calcium binding to TnC weakens
the binding of TnI and TnT to actin-TM in the region of Cys-190 of TM,
and TnT is required in the Tn complex for it to remain associated with
the thin filament (58-61; reviewed in (9) and (11) ). Troponin T, as well as the ternary Tn complex, greatly
increases the affinity of TM for actin, a consequence of tight
association between TnT and TM, as well as TnT and actin(62) .
The work presented here indicates that the striated-specific TM COOH
terminus, especially residues 258-275, is designed for strong,
isoform-specific interaction of Tn (presumably TnT) with TM on the
actin filament both in the presence and absence of
Ca
. Optimizing this interaction is important for thin
filament assembly, inhibition of the actomyosin ATPase in the absence
of Ca
, and full activation in the presence of
Ca
.