(Received for publication, October 3, 1995; and in revised form, December 11, 1995)
From the
Tropomyosin is a coiled-coil protein that binds along the length
of filamentous actin and contains sequence repeats that correspond to
actin monomers in the filament. Analysis of striated muscle
-tropomyosin mutants in which internal sequence has been deleted
or replaced with non-tropomyosin sequence showed that the following
parameters are important for high affinity, cooperative binding of
tropomyosin-troponin to actin. 1) Tropomyosin must be a coiled coil
along its entire length. 2) An integral number of repeats corresponding
to the actin monomers along its length is more important than the total
number. 3) In comparison, the actin affinity is relatively insensitive
to changes in the sequence of the internal regions of tropomyosin. The
results suggest that the internal sequence repeats function as weakly
interacting spacers to allow proper alignment of the ends on the
regulated actin filament.
Periodic patterns of amino acids are a common feature of fibrous
and structural proteins including collagen, tropomyosin (TM), ()spectrin, and nebulin. Certain periodicities are crucial
for formation of the basic molecular structure of the protein, as the
Gly-X-Y repeat is for the collagen triple helix (1) and the heptapeptide repeat of hydrophobic amino acids is
for the coiled coil(2, 3) . Some fibrous proteins
contain additional repeats that have been postulated to be related to
assembly of higher order structures, such as those in TM for actin
binding (4, 5, 6) and those in myosin for
filament assembly(7) . The present study addresses 1) the
importance of a 7-fold repeat and 2) the sequence requirements for
binding of striated muscle
-TM to the regulated actin filament
(actin with troponin (Tn)).
Tropomyosins form a family of
highly-conserved actin binding proteins found in virtually all
eucaryotic muscle and nonmuscle cells(8, 9) .
Tropomyosin is a two-chained, parallel coiled coil along its entire
length except for the ends whose structure is
unknown(10, 11) . It is localized in the long pitch
grooves of the helical actin filament(12, 13) .
Functions common to TMs are to bind cooperatively to F-actin, to
stabilize and stiffen the actin filament, and to allow cooperative
activation of the actin filament by
myosin(14, 15, 16, 17) . In striated
muscle, the TMTn complex regulates actin-myosin interaction in a
Ca
-dependent fashion(18, 19) .
The lengths of TM isoforms correspond to an integral number of actin
monomers in the filament: seven in 284-residue TMs found in muscle and
certain nonmuscle isoforms, six in 247-residue nonmuscle isoforms, and
five in yeast TMs(8, 9) . This relationship implies
the presence of periodic binding sites that correspond to the number of
actin monomers spanned by a single TM molecule on the actin filament.
McLachlan and Stewart (5) and Phillips (6) identified a
poorly-conserved 7-fold periodic repeat of amino acids in striated
-TM that is sufficiently regular to correspond to actin binding
sites. To test their hypothesis, we previously made a series of nested
deletions in the chicken striated
-TM cDNA in the region encoding
the second actin binding site (residues 47-88). The deletions
corresponded to one-half, two-thirds, and one actin binding site, based
on there being seven sites (repeats) per TM molecule(20) .
Analysis of these mutants showed that an integral 7-fold periodicity is
important for binding of TM
Tn to actin. Here we address, first,
whether the repeats function primarily as quasi-equivalent actin
binding sites or as weakly interacting spacers to ensure the proper
alignment of the ends (known to be important for actin binding)
relative to each other and to actin monomers on the regulated actin
filament. Second, we determined the structural requirements of the
repeats for cooperative binding of TM to the regulated thin filament.
The nucleotides encoding amino acid residues 89-123 (dAC3-35), 86-127 (dAC3-42), 47-123 (dAC23) and 47-60 (dAC2-14) were deleted using the following oligonucleotides (antisense sequence, the slash indicates the site of the deletion): dAC3-35, 5`-ACCTTCATTCCTCTTTC/CAGGGAAGCTACTTC-3`; dAC3-42, 5`-GCTCTATTTTCAATGACCTTT/ACTTCACTCTCAGC-3`; dAC23, 5`-GACCTTCATTCCTCTTTC/CAGAGCCACCAGC-3`; dAC2-14, 5`-ATCTTTAAGGGACTCGGA/CAGAGCCACCAG-3`. The dAC2 mutant was described in (20) . The replacement mutants were made using the following oligonucleotides (antisense sequence, the slash indicates the site of the deletion): 2zip, 5`-ATCTTTAAGGGACTCGGAAAGATGGTAGTTTTTAGAAAGAAGTTCTTCAACTTTGTCTCACAGAGCCACCAG-3`; 3zip, 5`-GCCAAGCGCTCCTGAAGATGGTAGTTTTTAGAAAGAAGTTCTTCAACTTTATCTTCCAGGGAAGCTAC-3`; 2rc, 5`-ATCTTTAAGGGACTCGGAGTAACCGTCACCTTTACGACCATCGCCTTCACGACCGTCGCCCAGAGCCACCAG-3`; 3rc, 5`-GCCAAGCGCTCCTGGTAACCGTCACCTTTACGACCATCGCCTTCACGACCGTCGCCCAGGGAAGCTAC-3`.
Chicken pectoral actin and Tn were purified as described previously (26, 27) .
The observed K values
differed depending on the ionic strength and other experimental
conditions employed in the course of the research. However, the order
of actin affinities of the TM variants remained constant over time and
at the two ionic strengths with the exception that dAC3-35 was
close to that of dAC2 at 150 mM, whereas in 200 mM NaCl, it was similar to dAC3-42. TMs from different
preparations had indistinguishable affinities.
The bound and free TM
were determined by quantitative densitometry of SDS-polyacrylamide gels
stained with Coomassie Blue(29, 30) . Bound TM was
expressed as the fraction of maximal binding, based on the TM/actin
ratio in the pellets. Free TM was determined by the analysis of TM in
the supernatant, using chicken pectoral -TM as a standard. The
parameters reported from curve fitting are in Table 1.
Protein concentrations were determined using a microbiuret assay with bovine serum albumin as a standard (31) or by determining the specific tyrosine absorbance(32) . Extinction coefficients (1% at 280) were used to calculate the concentration of actin (11.0) and Tn (4.5).
Figure 1:
Actin binding site deletion
mutants. A, design. Integral actin binding repeats were
deleted resulting in TMs that span five or six actin monomers on the
filament, versus seven in wild-type TM. The drawing shows one
TM and the actin monomers along its length in one strand of the helical
actin filament, each actin with a hypothetical TM binding site. The N
terminus of TM is at the left. The large and small circles relate to the - and
-actin binding
sites postulated by McLachlan and Stewart(5) . Troponin is not
illustrated. Both dAC2 and dAC3, deletion of site 2
or 3 respectively, give rise to TMs that should span six instead of
seven actin monomers. In dAC23, both sites 2 and 3 were
deleted, and the resulting TM should span five actin monomers. In the
filament, the TM molecules would be aligned head-to-tail along the
length of the filament. B, actin binding of
tropomyosin-troponin, with Ca
. Recombinant,
unacetylated TM was cosedimented with actin at 20 °C in 200 mM NaCl; 2 mM MgCl
; 0.2 mM CaCl
; 20 mM imidazole, pH 7.0; 0.5 mM dithiothreitol; 2.5 µM chicken pectoral actin;
chicken pectoral Tn and TM in a 1.2:1 molar ratio, 0 to 2-15
µM, depending on the TM, as described under
``Experimental Procedures.'' Bound TM is expressed as the
fraction of maximal binding, based on the TM/actin ratio in the
pellets. The parameters reported from curve fitting are in Table 1. Each curve is calculated from the data from two
independent experiments, except for WT, which is from three
experiments.
The mutants were expressed in E. coli to produce TMs
unacetylated at the N-terminal Met(35) . N-Acetylation
is required for striated -TM to bind tightly to actin, but in the
presence of Tn, unacetylated TM binds well and regulates the actomyosin
ATPase(28, 29, 35, 36) . We measured
the actin affinity of TM in a TM
Tn complex with saturating
Ca
. The data were analyzed using the Hill equation as
well as a linear lattice model(37, 38) . The K
values of both site 3 mutants for regulated
actin were indistinguishable, 2.7-fold weaker than dAC2, and almost
20-fold weaker than wild type (Fig. 1B; Table 1).
The mutant TMs were reduced in both affinity of TM
Tn for an
isolated site on actin (K
) and cooperativity
(
or y). The main effect of the dAC2 deletion
was reduced cooperativity. The dAC3 mutants had lower actin affinity
than dAC2 without further reduction in cooperativity, implying that
site 3, a highly conserved region of TM, is more critical for actin
binding than site 2.
If each repeat (site) contributes individually
to the overall actin affinity, then deletion of two sites should reduce
the actin affinity more than deletion of one site. In dAC23, 77 amino
acids corresponding to sites 2 and 3 were deleted (Fig. 1A, residues 47-123), close to the length
of two actin binding sites according to the McLachlan and Stewart model
(78 residues; (5) ), and the same as in the Phillips
proposal(6) . Surprisingly, the K of
dAC23 TM
Tn for actin was reduced only 2-fold compared with
wild-type TM, and the cooperativity was essentially unchanged. The K
of dAC23 was close to that of dAC2, considerably
higher than either dAC3 mutant, suggesting that the number of repeats
is not a major determinant of actin affinity.
The TM mutants of
different lengths typically saturated the actin filament at the same
TM/actin mass ratio. This implies that at saturation the TMTn
complexes are aligned head-to-tail along the actin filament,
independent of the length of the TM. Considered in terms of mass (versus the molecular weight of the TM), the K
of dAC23 is 62% of wild type (versus 46% when calculated in terms of molecular weight), while the K
is 93% of wild type (versus 67%). The
TnI/TM ratio at saturation was also proportional to the length of the
TM, consistent with one Tn binding site/TM on the thin filament.
The
high affinity and cooperativity of dAC23 relative to the dAC2 or dAC3
mutants suggests that individual repeats (sites) contribute little to
the affinity of TMTn for actin and that they function primarily
as weakly interacting spacers. The three single-site deletion mutants
bound with lower cooperativity than either dAC23 or wild type. Since
the single site deletions do not correspond perfectly to one-seventh of
TM (39 residues), the ends may be mismatched relative to each other and
to the actin monomer in the filament. This misalignment, if propagated,
may result in a long range disorder that could reduce the cooperativity
and affinity of binding.
Tropomyosin binding to actin has traditionally been modeled in terms of one TM molecule and the actin subunits along its length in one strand of the actin filament with cooperativity primarily attributed to direct or indirect end-to-end associations between adjacent TM molecules on the filament(37, 38) . An alternative interpretation is that when the TM is perfectly matched to the filament and the ends are properly aligned with respect to each other and the monomers in the actin filament, the TM molecules may form a seamless cable with, for the purposes of this analysis, an infinite number of equivalent and indistinguishable sites. We consider this model unlikely because it is well established that alterations at either the N or C terminus can profoundly affect cooperative actin binding (28, 29, 35, 36, 39-46, 50). Furthermore, as will now be discussed, changes in internal sequence have small, but significant effects on actin binding.
To investigate the structural requirements for actin binding in a more fundamental sense, we replaced 14 residues of site 2 (residues 47-60) or site 3 (residues 89-102) with 14 residues of the GCN4 leucine zipper (2zip, 3zip; (49) ) or a random coil sequence (2rc, 3rc; Fig. 2A). The GCN4 (zip) sequence shares only one amino acid identity with site 2 and none with site 3 (Fig. 2C). Since the sequence is highly favorable for coiled coil formation, the length and conformation of the 2zip and 3zip should be similar, if not identical, to wild type TM. The random coil sequence (Fig. 2C) was designed with glycines at every second or third residue to prevent formation of secondary structure. The rest of the amino acids were charged in keeping with the highly charged nature of TM. The random coil replacement would interrupt the coiled coil structure of TM. All replacement mutants were expressed well in E. coli and could be purified using the normal procedure with the exception of the 3rc, which was not expressed at detectable levels using two different vectors in two different E. coli strains. The 2zip and 3zip mutants were similar in stability to wild type, whereas 2rc was less stable (See ``Experimental Procedures'' and (25) ).
Figure 2:
Replacement of tropomyosin residues with
leucine zipper and random coil sequences. A, design. The
format of the diagram is described in Fig. 1A. Here, 14
residues of site 2 or site 3 were replaced with 14 residues of a GCN4
leucine zipper sequence ((47) , 2zip, 3zip)
or 14 residues that should not form an ordered secondary structure (2rc, 3rc). B, actin binding of
tropomyosin-troponin, with Ca. The conditions are the
same as in Fig. 1B. dAC2-14 is a mutant in which
14 residues of site 2 (residues 47-60) were deleted. The TM actin
binding sites would be out of register with the TM binding sites on
actin. The 3rc mutant was not expressed in E. coli. The
observed binding by 2rc and dAC2-14 is nonsaturating and at the
level of trapping. The number of independent data sets used to
calculate each curve is as follows: wild type (WT), 3; 2zip, 2; 3zip, 3; 2rc, 1; dAC2-14, 1. The experiments for 2rc and dAC2-14
were carried out in the above buffer with 150 mM NaCl in an
attempt to increase actin affinity. The affinity of 2rc for regulated
actin was also undetectable at 4 °C. C, amino acid
sequences of replaced regions and comparison with the tropomyosin
sequence. Lower case letters refer to the heptapeptide repeat
of amino acids in the coiled coil; a and d are at the
interface between the two
-helices(3) . Asterisks indicate identities.
Both 2zip and 3zip bound well to regulated actin
with affinities similar to that of dAC2 (Fig. 2B, Table 1), an effect that is in the same order of magnitude as
switching exon 2 or 6 in different TM
isoforms(43, 45, 47, 48) .
Interestingly, both the actin affinity and the cooperativity were
reduced. Since the lengths of the 2zip and 3zip should be the same as
wild type, the relationship of the ends to each other and to actin
should not be altered. This implies that the change in cooperativity
relates to the interaction of TMTn with actin or to the local
sequence and conformation of the TM molecule. It is also possible that
the mutation has a long range effect on the ends.
In contrast, 2rc
had no detectable affinity for regulated actin in the presence or
absence of Ca, even at 4 °C, where it was nearly
fully helical (Fig. 2B). The 2rc chains are parallel
since they could be fully cross-linked via Cys-190. A second mutant
where the first 11 residues of the random coil sequence replaced
residues 39-49 of site 2 also showed no detectable actin
affinity. Fig. 2B shows in addition that deletion of 14
residues of site 2 (dAC2-14) corresponding to one third of a
repeat, resulted in loss of actin affinity as predicted by previous
work(20) .