(Received for publication, January 26, 1995; and in revised form, August 21, 1995)
From the
Dictyostelium myosin deficient in the essential light chain
(ELC) does not function normally either in vivo or in
vitro (Pollenz, R. S., Chen, T. L., Trivinos-Lagos, L., and
Chisholm, R. L.(1992) Cell 69, 951-962). Since normal
[Medline]
myosin function requires association of ELC, we investigated the
domains of ELC that are necessary for binding to the myosin heavy chain
(MHC). Deleting the NH-terminal 11 or 28 amino acid
residues (
N11 or
N28) or the COOH-terminal 15 amino acid
residues (
C15) abolished binding of the ELC to the MHC when the
mutants were expressed in wild-type (WT) cells. In contrast, the ELC
carrying deletion or insertion of four amino acid residues (D4 or I4)
in the central linker segment bound the MHC in WT cells, although less
efficient competition with WT ELC suggested that the affinity for the
MHC is reduced. When these mutants were expressed in ELC-minus
(mlcE
) cells, where the binding to the heavy chain is
not dependent on efficient competition with the endogenous ELC,
N28 and
N11 bound to the MHC at 15% of WT levels and
C15
did not bind to a significant degree. I4 and D4, however, bound with
normal stoichiometry. These data indicate that residues at both termini
of the ELC are required for association with the MHC, while the central
linker domain appears to be less critical for binding. When the mutants
were analyzed for their ability to complement the cytokinesis defect
displayed by mlcE
cells, a correlation to the level
of ELC carried by the MHC was observed, indicating that a
stoichiometric ELC-MHC association is necessary for normal myosin
function in vivo.
Myosin is a mechanochemical enzyme that generates force during
muscle contraction and has a fundamental role in a variety of cell
movements (for review, see (2) ). Conventional myosin is a
hexameric protein composed of a pair of heavy chains (MHC) ()and two pairs of light chains, called essential light
chains (ELC) and regulatory light chains (RLC). Both light chains bind
to the neck of myosin(3, 4) . We have shown that
ELC-deficient Dictyostelium cell lines are defective in
cytokinesis, and myosin purified from these cells does not show
significant actin-activated ATPase activity(1) . Since normal
myosin function requires association of the ELC, we set out to map the
domains of the ELC that are critical for ELC-MHC interaction.
Sequence comparisons of ELCs from various sources show that the
COOH-terminal half of the molecule is more conserved than the
NH-terminal half (5, 6) and that it may
represent the site required for association with MHC. Skeletal muscle
alkali light chain A1 in which the COOH-terminal 14 residues were
chemically removed did not bind the S1 heavy chain, although the
conformation remained largely unchanged(7) . Transfection
studies also showed that a COOH-terminal truncation of the alkali light
chain A1 failed to colocalize with acto-myosin structures in cultured
myocytes (8) .
The ELC belongs to the calmodulin-troponin
gene family, which contains four helix-loop-helix structures, also
known as EF-hands (for review, see (9) ). The atomic structures
of calmodulin (CaM) and troponin C show two terminal globular lobes
linked by a central helix, with each lobe containing a pair of
EF-hands(10, 11) . Although ELCs may no longer bind
Ca due to amino acid deletions or substitutions in
the Ca
-coordinating sites(9) , several lines
of evidence suggest that the overall structure has been preserved. The
ELC has many physical and chemical properties similar to those of CaM
and troponin C, including Stokes radius, molecular weight,
sedimentation coefficient, and radius of gyration(4) .
Secondary and tertiary structural modeling, based on factors thought to
control protein folding, also predicted similar tertiary arrangements
for ELC, troponin C, and CaM(12) . Recently, three-dimensional
structures of myosin S1 (13) and the regulatory domain of
scallop myosin (14) confirmed these predictions. Based on these
structures, we divided the Dictyostelium ELC into three
structural domains: an NH
-terminal globular domain, a COOH
globular domain, and a central linker domain. The participation of
these domains in ELC-MHC interaction was assessed. In vitro mutagenesis was used to modify the Dictyostelium ELC, and
the altered ELCs were expressed in both WT and mlcE
cells. The ability of mutant ELC to associate with the MHC in
vivo and to rescue the phenotypic defects of
mlcE
cells was analyzed.
Figure 1:
Expression and
association of the mycELC in WT cells. Protein samples from a mycELC transformant were run on 15% SDS-polyacrylamide gel,
transferred to nitrocellulose, and probed with myosin polyclonal
anti-serum NU 48. Lane 1, whole cell lysate of 1
10
cells; lane 2, Triton-insoluble cytoskeletons; lane 3, ATP-released myosin supernatant; lane 4,
purified myosin. The arrowhead points to the mycELC.
Figure 2: Structural representations of terminal deletion mutants. Positions of four helix-loop-helix structures of the Dictyostelium ELC (Dd ELC) are illustrated based on sequence alignment with the Dictyostelium CaM. Solid bars (denoted from A to H) represent helices, and curved lines represent loops. The myc-epitope tag is indicated.
Figure 3:
Expressed terminal deletion mutants do not
associate with MHC in WT cells. Transformants from N28,
N11,
and
C15 were analyzed by Western blots as in Fig. 1. Lanes 1, 3, and 5, whole cell lysates; lanes 2, 4, and 6, Triton-insoluble
cytoskeletons. Arrowheads point to the
mutants.
The involvement of the COOH terminus of
the ELC in heavy chain binding is consistent with previous
observations(7, 8) , but participation of residues at
NH terminus of the ELC has not been previously
demonstrated. We tested the possibility that the
NH
-terminal residues are required for maintaining an
overall conformation of the ELC rather than being in a direct
association with the heavy chain. Amino acid residue 15, a
phenylalanine residue that is conserved among ELCs(28) , was
replaced by an alanine. This residue corresponds to Phe-19 of CaM,
which has five contacts in the CaM
M13 peptide
complex(29) . Fig. 4shows that the F15A ELC mutant was
expressed to levels comparable with the mycELC (lane
1) and partitioned in the cytoskeletal fractions (lanes 2 and 3), suggesting that it is associated with the MHC.
But unlike the mycELC transformants (Fig. 1), the
endogenous ELC appeared in the cytoskeletal fractions of the F15A
transformants (lanes 2 and 3), suggesting that the
F15A ELC had reduced ability to compete with the endogenous ELC for the
binding to the MHC. Quantification of MHC-bound ELC showed that 85% of
the MHC carried the F15A ELC in WT cells (Table 1). The reduced
binding affinity of this substitution mutant supports the idea that
residues at the NH
terminus of ELC associate directly with
the heavy chain.
Figure 4: Expression and association of the F15A mutant in WT cells. One of the F15A trasformants was analyzed by Western blot as in Fig. 1. Lane 1, whole cell lysate; lane 2, Triton-insoluble cytoskeletons; lane 3, ATP-released myosin supernatant.
While this work was in progress, mlcE cell lines produced by gene targeting became available. (
)The ELC mutants were expressed in mlcE
cells to assess their binding to MHC in the absence of competing
endogenous ELC. While the levels of expression were comparable with
those seen in WT cells,
N28 and
N11 bound to MHC at 15% of WT
levels, whereas
C15 did not bind to any significant extent. F15A
showed binding comparable with WT ELC. Since F15A was at least 10-fold
overexpressed, its reduced binding affinity was apparently masked by
its overexpression in mlcE
cells.
Despite the fact that both I4 and D4 were expressed to a level comparable with that of mycELC in WT cells, they did not compete as efficiently for the MHC binding as the mycELC (Fig. 5). These two mutants bound to MHC at 40% of the mycELC level in WT cells (Table 1), indicating that the ELC carrying deletion or insertion of residues in the central domain bind MHC with reduced affinity.
Figure 5: Association of I4 and D4 with MHC in WT cells. The ATP-released myosin supernatants from I4 and D4 transformants were analyzed by Western blot as in Fig. 1. The ATP-released myosin supernatant from mycELC transformants is used as control. Arrowheads indicate mycELC, I4, and D4, respectively.
When the I4 and D4 mutants
were overexpressed in mlcE cells, they associated
with MHC stoichiometrically, a situation similar to the F15A ELC. To
test the importance of the linker domain in myosin enzymatic function,
myosin containing I4 and D4 was purified and assayed for ATPase
activity and the ability to support movement of actin filaments in an in vitro motility assay. As shown in Table 2, both
myosins had WT activities.
To determine the biochemical properties
of a ``weak binding'' mutant, myosin was purified from cells
expressing N11. In purified myosin preparations,
N11 bound to
MHC at 5% of WT levels. Similar to ELC-minus myosin,
N11 myosin
did not exhibit significant actin-ATPase and failed to move actin
filaments in vitro (Table 2).
Figure 6:
The abilities of ELC mutants to rescue the
cytokinesis defect of mlcE cells. Cells transformed
with ELC mutants were grown in suspension for 3 days and taken for
nuclei staining by DAPI. More than 100 cells were scored for each
strain. The numbers are the average of two or three independent
experiments. Solid bars represent the efficiency of
cytokinesis, and stippled bars represent the levels of mutant
ELCs associated with the heavy chain.
It has been reported that myosin lacking
the RLCs has a tendency to
aggregate(33, 34, 35) , presumably mediated
by the exposed, hydrophobic -helix that serves as binding sites
for myosin light chains(13, 14) . An important
question is whether the poor cellular myosin function observed with the
weak binding mutants
N28,
N11,
C15 is due to a defect in
enzymatic activity or aggregation of a significant amount of the myosin
lacking bound ELC. To address this question, crude cellular extracts of
mlcE
and WT cells were subjected to gel filtration to
compare the association state of the myosin. As shown in Fig. 7,
the ELC-minus myosin fractionated with the same pattern as wild-type,
suggesting there are no gross differences in the physical state of
myosin in the two cells. Thus the multinucleate phenotype associated
with the weak binding mutants seems unlikely to result from abnormal
myosin aggregation.
Figure 7:
Gel filtration profiles of myosin
cytoskeletons from mlcE and WT cells. High salt
cytoskeletons obtained from mlcE
and WT cells were
fractionated on Sephacryl-500 column. Elution of myosin was monitored
by measuring the densities of MHC in eluted
fractions.
The structure of CaMM13 peptide complex shows that
residues in both lobes of calmodulin are in contact with the M13
peptide (a 26-amino acid peptide containing the CaM binding domain of
myosin light chain kinase); residues in the helices account for the
vast majority of those contacts(29, 36) . By analogy
to this structure, the two terminal helices A and H of the Dictyostelium ELC were deleted. The deletions resulted in a
loss of ELC binding to the MHC when the mutants were expressed in WT
cells. Since the atomic structure of CaM shows independent folding of
the two lobes and no interactions between them, it is unlikely that
mutations in one lobe affect the folding or tertiary structure of the
other. If this is true with ELC, then the deletion results suggest that
residues in both the COOH-terminal lobe and NH
-terminal
lobe are important for association with the heavy chain. The
participation of the NH
terminus is further supported by
the substitution mutant F15A, in which a phenylalanine to alanine
substitution resulted in reduced binding of the ELC to the MHC. It is
conceivable, therefore, that ELC may bind MHC with both lobes in a
manner similar to that of CaM binding its substrates. The recently
published atomic structures of myosin S1 (13) and the
regulatory domain of scallop myosin (14) are consistent with
this idea. Our results further indicate that neither lobe is sufficient
and that both are necessary for the ELC-MHC association. When expressed
in mlcE
cells,
C15 did not show significant
binding to the MHC, whereas
N11 and
N28 retained low levels
of association. It is possible that the COOH terminus of the ELC may
contain a major binding site for the heavy chain, and the binding is
strengthened by the association with the NH
terminus. A
similar binding mode has been observed for smooth muscle regulatory
light chain(37) .
Similar to the CaMM13 peptide
complex, the major contacts between ELC and MHC are
hydrophobic(13, 14) . Phenylalanine 15, a conserved
hydrophobic residue, is shown in this study to take part in the
process. There are in total 204 contacts observed between the ELC and
MHC(14) , implying that many residues of the ELC participate in
the ELC-MHC association. A single amino acid substitution, therefore,
might not be expected to produce a dramatic effect on binding, as
observed for the F15A mutant, which caused a modest reduction in the
binding affinity for the heavy chain.
The ELC has a dumbbell-like fold similar to that of CaM(13, 14) . Like the bound form of CaM, the helix in the linker region of ELC is unwound to facilitate binding of the two lobes on the heavy chain. It is likely, therefore, that deletion or insertion of residues in the linker region will lead to suboptimal contacts of the terminal domains with respect to the binding site on the heavy chain. This seems likely to provide a structural basis for the reduced binding affinity seen with the I4 and D4 mutations. The D4 mutant, in which only six out of 10 residues in the linker region are retained, still bound the MHC, albeit with a reduced affinity, suggesting that the linker segment must be flexible enough to tolerate significant shortening. In addition, the deletion or insertion of four residues in the linker domain does not seem to affect myosin enzymatic function as assayed by ATPase activity measurements and in vitro motility assays. Similar results were observed in mutagenesis studies of CaM, where it has been proposed that the linker domain functions as a flexible structure to accommodate binding of the two lobes to a range of substrates(31, 38, 39) . It is likely that the linker region of ELC may also function primarily as a structural element to place the two globular domains in an optimal contact with the heavy chain.
It has been suggested that one function of the
myosin light chains is to provide structural support for the region of
single -helix of the heavy chain that exists between the head and
the coiled-coil tail domain. One notion is that by stiffening the
myosin neck, the light chains contribute to an extension of the
effective power stroke resulting from conformational changes in the
head(13, 33, 40) . If this is the case, then
the I4 mutant, which extends the central linker domain but apparently
has normal myosin function, must still provide adequate structural
support. It would be interesting to determine how large a central
linker region could become before it would no longer provide sufficient
structural support.
The light chain binding region of the heavy
chain, a long -helix, is hydrophobic in nature and shielded from
solvent by association with light chains(13, 14) .
Thus, one apparent function of light chains is the stabilization of
this structure. Consistent with this structural analysis, myosins
lacking the RLCs have a tendency to aggregate through the neck
region(33, 34, 35) . Our results show that
the ability of ELC mutants to rescue the cytokinesis defect of
mlcE
cells correlates with their ability to bind to
heavy chain, establishing that a stoichiometric binding of ELC is
necessary in vivo for normal myosin function. On the other
hand, the results from mutants F15A, I4, and D4 seem to suggest that as
long as the stoichiometric association between ELC and MHC is
maintained, myosin would be sufficiently functional to rescue the
defects seen in the ELC null mutant. In addition, the similarity of gel
filtration profiles of WT and mlcE
cells suggests
that the defects observed in mlcE
myosin are unlikely
to be due to abnormal aggregation of mlcE
myosin.
This raises the question of whether the ELC merely serves a structural
support role for the neck of myosin, or might it have other functions?
ELC binds to the heavy chain between the active site and the regulatory
light chain(13) . It is possible that it may also play a role
in modulating myosin enzymatic function and/or in transmitting
regulatory signals from the regulatory light chain to the active
center. With the atomic structures now in hand, further mutagenesis to
explore other functions of ELC are in progress.
In summary, we have
shown that residues at both termini of the ELC are necessary for the
ELC-MHC association. These data provide direct experimental support for
the structural models presented recently(13, 14) .
Residues in the central linker domain do not appear to be essential for
either ELC's binding or function, but they do seem to be required
for the optimal association of the two terminal domains on the
-helix of heavy chain. Furthermore, a stoichiometric ELC-MHC
association is necessary for normal myosin-dependent function in
vivo.
To whom correspondence should be addressed. Tel.: 312-503-4151; Fax: 312-503-5994.