Familial Hypertrophic Cardiomyopathy Mutations in the Regulatory
Light Chains of Myosin Affect Their Structure, Ca2+
Binding, and Phosphorylation*
Danuta
Szczesna
§,
Debalina
Ghosh
,
Qi
Li
,
Aldrin V.
Gomes
,
Georgianna
Guzman
,
Carlos
Arana
,
Gang
Zhi¶,
James T.
Stull¶, and
James D.
Potter
From the
Department of Molecular and Cellular
Pharmacology, University of Miami School of Medicine, Miami,
Florida 33136 and the ¶ Department of Physiology, University of
Texas Southwestern Medical Center, Dallas, Texas 75390
Received for publication, October 27, 2000, and in revised form, November 22, 2000
 |
ABSTRACT |
The effect of the familial hypertrophic
cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in
the regulatory light chains of human cardiac myosin has been
investigated. The results demonstrate that E22K and R58Q, located in
the immediate extension of the helices flanking the regulatory light
chain Ca2+ binding site, had dramatically altered
Ca2+ binding properties. The KCa
value for E22K was decreased by ~17-fold compared with the wild-type
light chain, and the R58Q mutant did not bind Ca2+.
Interestingly, Ca2+ binding to the R58Q mutant was restored
upon phosphorylation, whereas the E22K mutant could not be
phosphorylated. In addition, the
-helical content of phosphorylated
R58Q greatly increased with Ca2+ binding. The A13T
mutation, located near the phosphorylation site (Ser-15) of the human
cardiac regulatory light chain, had 3-fold lower
KCa than wild-type light chain, whereas
phosphorylation of this mutant increased the Ca2+ affinity
6-fold. Whereas phosphorylation of wild-type light chain decreased its
Ca2+ affinity, the opposite was true for A13T. The
-helical content of the A13T mutant returned to the level of
wild-type light chain upon phosphorylation. The phosphorylation and
Ca2+ binding properties of the regulatory light chain of
human cardiac myosin are important for physiological function, and
alteration any of these could contribute to the development of
hypertrophic cardiomyopathy.
 |
INTRODUCTION |
There is substantial evidence that myosin regulatory light chains
(RLC)1 play a primary
regulatory role in scallop and smooth muscle contraction, but
their functional role in mammalian striated (skeletal and cardiac)
muscle contraction is unclear. RLC, together with the essential light
chain, stabilizes the 8.5-nm-long
-helical neck of the myosin head,
with the N terminus of RLC wrapped around the heavy chain (1). Smooth
muscle contraction is initiated by RLC phosphorylation with a
Ca2+-calmodulin-activated myosin light chain kinase (MLCK)
(2, 3). However, in skeletal and cardiac muscle, RLC phosphorylation does not activate contraction but appears to play a modulatory role
(4). It was shown that RLC phosphorylation increased the Ca2+ sensitivity of force in skinned skeletal (5-7) and
cardiac (8) muscle fibers. In the human heart, several RLC isoforms are
expressed (9, 10) preferentially in the atrium and in the ventricle. Recent studies have revealed that the ventricular RLC is one of the
sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (11, 12). FHC is an autosomal dominant disease,
characterized by left ventricular hypertrophy, myofibrillar disarray,
and sudden death. It is caused by missense mutations in various genes
that encode for
-myosin heavy chain (13), myosin-binding protein C
(14), ventricular RLC and essential light chain (11, 12, 15), troponin
T (16), troponin I (17),
-tropomyosin (18), actin (19), and titin
(20). Depending on the affected gene, and the site of the mutation, FHC
has variable presentation with regard to its degree and severity and
the extent of myocardial disarray. The clinical manifestations of FHC
range from benign to severe heart failure and to sudden cardiac death.
The best characterized clinical cases include patients with
-myosin
heavy chain mutations who have a high level of cardiac hypertrophy and
those with troponin T mutations who have less hypertrophy, but a higher
incidence of sudden cardiac death in young adults. The first three
identified mutations in the RLC (A13T, E22K, and P95A) were shown to be
associated with a particular subtype of cardiac hypertrophy defined by
mid-left ventricular obstruction (11). Two other RLC mutations (F18L
and R58Q), identified by Flavigny et al. (12), were
associated with a typical form of hypertrophic cardiomyopathy, which
causes increased left ventricular wall thickness and abnormal
electrocardiograph findings with no mid-cavity obliteration.
The three-dimensional structure of the RLC demonstrates the close
proximity of FHC mutations to either the phosphorylation site of RLC
(Ser-15) or the Ca2+ binding site (amino acids 37-48).
Because of this distinctive arrangement of the FHC mutations within the
RLC, it was of interest to study their effect on the phosphorylation
properties of human cardiac RLC (HCRLC) as well as their effect on
Ca2+ binding. We have also investigated how these FHC
mutations influence the secondary structure of the HCRLC as well as the
combined effects of phosphorylation and Ca2+ binding on
their structure. We demonstrate that both processes, phosphorylation
and Ca2+ binding, are significantly altered by the FHC
mutations and their effect depends upon the specific location of the
missense mutation. The alterations in contractility that would result
from these mutations are not known at present, and it is therefore
not possible to know precisely how they might trigger the
hypertrophic process. It is likely, however, that such a response would
be part of a direct compensatory and/or adaptive mechanism of the heart
to maintain normal cardiac function. Assessing the mechanism by which FHC mutations alter RLC function will lead to a better understanding of
the physiological role of RLC in the regulation of cardiac muscle contraction.
 |
MATERIALS AND METHODS |
Mutation, Expression, and Purification of Wild-type HCRLC and the
FHC Mutants--
The cDNA for wild-type HCRLC was cloned by
reverse transcription-polymerase chain reaction using primers based on
the published cDNA sequence (GenBankTM accession no. AF020768) and
standard methods (21). The FHC RLC mutants: A13T, F18L, E22K, R58Q, and P95A, were generated using overlapping sequential polymerase chain reaction (21). Wild-type and mutant cDNAs were constructed with an
NcoI site at the N-terminal ATG and a BamHI site
following the stop codon to facilitate ligation into the
NcoI-BamHI cloning site of the pET-3d (Novagen)
plasmid vector and transformation into DH5
cloning host bacteria for
expression of the cDNAs of the wild-type HCRLC and the FHC mutants.
The cDNAs of the proteins were transformed into BL21 expression
host cells and protein expressed in large (16 liters) cultures.
Expressed proteins were purified using a Q-Sepharose column followed by
a DE-52 column, both equilibrated with 2 M urea, 25 mM Tris-HCl, 0.1 mM PMSF, 1 mM
dithiothreitol, 0.001% NaN3, pH 7.5. The proteins were
eluted with a 1000-ml salt gradient of 0-300 mM KCl. The
elution profiles from both the Q-Sepharose and the DE-52 columns for
wild-type (WT) HCRLC and A13T occurred in the range of 160-200
mM KCl, for F18L, R58Q, and P95A between 210-250
mM KCl, and for E22K between 230 and 280 mM
KCl. The final purity of the proteins was tested using 15%
SDS-PAGE.
Phosphorylation of Wild-type HCRLC and Its FHC Mutants--
The
proteins were phosphorylated with Ca2+-calmodulin activated
myosin light chain kinase (MLCK) in a solution containing: 50 µM protein (in 20 mM imidazole, 50 mM NaCl, 0.1 mM PMSF, pH 7.5), 0.5 µM MLCK, 5.0 µM calmodulin, 0.1 mM CaCl2, 12 mM MgCl2
and 5 mM ATP. A catalytically active truncated fragment of
the rabbit skeletal muscle MLCK was used in this study (22). The MLCK, missing the first 256 amino acids, was expressed in Sf9 cells infected by a recombinant virus (23). The phosphorylation reaction was
carried out for 2 h at room temperature or overnight on ice. After
phosphorylation the proteins were purified using a Q-Sepharose column
(conditions as described above). The level of phosphorylation was
measured using 8% urea-PAGE (24).
Flow Dialysis--
Flow dialysis was performed in a solution of
100 mM KCl, 20 mM imidazole, pH 7.0 (22 °C).
All proteins were equilibrated in this buffer prior to the
measurements. The flow dialysis experiments were performed according to
Colowick et al. (25) with modifications. Briefly, the upper
chamber of the apparatus containing the protein and the labeled
substrate (45Ca2+) was separated by a membrane
from the lower chamber. The buffer was pumped through the lower chamber
at a constant rate of 1.5 ml/30 s. The upper chamber was first
equilibrated with 0.4 ml of buffer for 15 min, followed by the protein
(0.4 ml) for 5 min. After adding 45Ca2+, an
equilibrium was attained by flowing buffer through the lower chamber
for 5 min. After steady state was reached, unlabeled substrate (Ca2+) was added at regular intervals and in varying
concentrations. Fractions were collected every 30 s, and the
effluent was sampled for measurement of radioactivity. The specific
radioactivity of 45Ca2+ used in the experiment
was 12-16 mCi/mg (from PerkinElmer Life Sciences), and 2 µCi
of 45Ca2+ per experiment gave sufficient
radioactivity in the dialysate for accurate measurements. Data were
presented using Scatchard analysis (26, 27).
|
(Eq. 1)
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CCa-bound and
CCa-free represent the concentration of the
bound and free metal, respectively, Cp is the
concentration of the protein, n is the total number of
Ca2+ binding sites, and KCa is the
Ca2+ binding affinity.
CD Measurements--
Far-UV circular dichroism spectra (CD) were
obtained using a 1-mm path quartz cell in a Jasco J-720
spectropolarimeter. Spectra were recorded at 195-250 nm with a
bandwidth of 1 nm at a speed of 50 nm/min and a resolution of 0.2 nm.
Analysis and processing of data were done using the Jasco system
software (Windows Standard Analysis, version 1.20). Ten scans were
averaged, base lines subtracted, and no numerical smoothing applied.
Mean residue ellipticity ([
]MRE, in
degrees·cm2/dmol) for the spectra were calculated
(utilizing the same Jasco system software) using the following equation
(28-30).
|
(Eq. 2)
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[
] is the measured ellipticity in millidegrees,
Cr is the mean residue molar concentration, and l
is the path length in cm. The optical activity of the buffer with or
without Ca2+ was subtracted from relevant protein spectra.
The
-helical content for each protein was calculated using the
standard equation for [
] at 222 nm (31).
|
(Eq. 3)
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fH is the fraction of
-helical content
(fH × 100, expressed in %). The measurements
were performed at 22 °C, in 10 mM phosphate buffer, 60 mM NaF, and 1 mM EGTA or 0.1 mM
CaCl2 at pH 7.0. NaF was used to avoid the strong
absorption of chloride ions in the far ultraviolet (32). The protein
samples were equilibrated with respective Ca2+ buffers
before the measurements. Spectra are presented as the mean residue ellipticity.
 |
RESULTS |
To study the effects of FHC mutations on the phosphorylation,
Ca2+ binding and the secondary structure of HCRLC, we have
cloned and expressed the WT and five FHC mutants of HCRLC: A13T, F18L, E22K, R58Q, and P95A. Fig. 1A
presents the amino acid sequence of human ventricular RLC and all the
known specific missense mutations that have been associated with FHC.
The Ca2+ binding (residues 37-48) and the phosphorylation
sites (Ser-15) are also illustrated. Fig. 1B demonstrates
the three-dimensional representation of the HCRLC derived from the
crystal structure of chicken skeletal myosin S1 (1). The N-terminal
region of HCRLC containing the phosphorylation site and two of the FHC
mutations, A13T and F18L, is not shown since this region of the RLC was
unresolved in the reported crystal structure (1). The three-dimensional representation of the HCRLC (Fig. 1 B) suggests that the mutations A13T, F18L, E22K, and P95A are located in close proximity to the phosphorylation site of RLC (Ser-15), whereas the E22K and R58Q mutations occur in the immediate extension of the helices flanking the
Ca2+ binding loop.

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Fig. 1.
Localization of the FHC mutations in
HCRLC. A, the amino acid sequence of HCRLC illustrating
FHC mutations: A13T, F18L, E22K, R58Q, and P95A; Ca2+
binding (residues 37-48) and the phosphorylation (Ser-15) sites.
B, the three-dimensional representation of HCRLC with
illustrated FHC mutations and the Ca2+ binding loop
(Swiss-Prot accession number Q14908).
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Ca2+ Binding to HCRLC and Its FHC
Mutants--
HCRLC-WT and the FHC mutants were tested for
Ca2+ binding using the flow dialysis method. As shown in
Fig. 2 and Table
I, HCRLC-WT bound Ca2+ with
relatively low affinity, KCa = 6.67 ± 0.21 × 105 M
1
(average of n = 3 flow dialysis experiments ± S.D.). The Scatchard plots presented here are representative of one
flow dialysis experiment, while the affinity constants demonstrated in
Table I are the average of 2-4 flow dialysis experiments ± S.D..
Three of the FHC mutations: A13T, F18L, and P95A, decreased their
Ca2+ binding affinity ~3-fold compared with HCRLC-WT
(Table I, n = 3, p < 0.003). However,
two other FHC mutants, E22K and R58Q, dramatically changed the RLC's
Ca2+ binding properties. The KCa
value decreased by ~17-fold for the E22K mutant (Fig. 2 and Table I,
n = 3, p < 0.001), whereas the R58Q
mutation completely impaired Ca2+ binding (Table I).

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Fig. 2.
Ca2+ binding to HCRLC and its FHC
mutants. Representative Scatchard plots for HCRLC-WT, A13T, and
E22K. The KCa values for HCRLC-WT and all of the
FHC mutants are listed in Table I. Flow dialysis was performed in the
solution of 100 mM KCl, 20 mM imidazole, pH
7.0, and 2 µCi of 45Ca2+/experiment.
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Table I
Effect of the FHC mutations and phosphorylation on the Ca2+
binding to human cardiac RLC
Data are the average of n = 3 flow dialysis
experiments ± S.D. ND, not determined.
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Effect of Phosphorylation--
Phosphorylation of HCRLC-WT with
Ca2+-calmodulin activated MLCK decreased its
Ca2+ binding affinity by 7.4-fold
(KCa = 0.90 ± 0.06 × 105
M
1, Fig.
3A and Table I,
n = 3, p < 0.001). However,
phosphorylation of the FHC mutants affected their Ca2+
binding properties in various ways (Figs. 3 and
4). The most dramatic effect was observed
for the E22K mutant. This substitution prevented the E22K mutant from
becoming phosphorylated. Even a 20-fold increase of the
enzyme/substrate ratio and longer incubation time did not result in
phosphorylated E22K (Fig. 4, lane 5). As shown in
Fig. 4, the gel migration of the E22K mutant was slower than HCRLC-WT,
F18L, and R58Q mutants, due to the Glu
Lys replacement that
resulted in an additional positive charge of the E22K protein. The E22K
mutation changes the isoelectric point of HCRLC from pI = 4.92 to
pI = 5.10; therefore, the nonphosphorylated E22K migrates slower
than the nonphosphorylated wild-type HCRLC. Accordingly, the
phosphorylated forms of HCRLC-WT and its FHC mutants migrated faster
than the nonphosphorylated ones due to the acidic phosphate group
attached to Ser-15 of the phosphorylated proteins (Fig. 4). A large
effect of phosphorylation was also observed for the A13T mutation. As
shown in Fig. 1, this mutation is located next to Ser-15, the
phosphorylation site of HCRLC. Phosphorylation of A13T resulted in a
large increase (KCa value increased from 2.06 ± 0.23 × 105
M
1 to 1.33 ± 0.02 × 106 M
1) in its
Ca2+ binding affinity compared with nonphosphorylated A13T
(Fig. 3B and Table I, n = 3, p = 0.016). Interestingly, phosphorylated A13T
demonstrated a 15-fold greater affinity for Ca2+ than
phosphorylated HCRLC-WT, whereas nonphosphorylated A13T bound
Ca2+ with a 3-fold lower affinity than
nonphosphorylated-WT. No effect of phosphorylation on Ca2+
binding to F18L was observed (Table I, n = 3). However,
there was an interesting effect of phosphorylation on Ca2+
binding to the R58Q mutant. This mutant did not bind Ca2+
in the nonphosphorylated state but did bind Ca2+ when
phosphorylated (KCa = 3.04 ± 1.02 × 105 M
1) (Table I).

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Fig. 3.
Effect of phosphorylation on Ca2+
binding to HCRLC-WT and the A13T mutant. Flow dialysis method was
utilized to determine KCa of HCRLC-WT and its
FHC mutants (see Table I and Fig. 2). Data were analyzed using
Scatchard plot:
CCa-bound/CCa-free/Cp = KCa·CCa-bound/Cp + n·KCa, where
CCa-bound and CCa-free
represent the concentration of the bound and free Ca2+,
respectively; n is the number of the total Ca2+
binding sites of the protein; and KCa is the
Ca2+ binding affinity constant.
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Fig. 4.
8% urea-PAGE of HCRLC-WT and its FHC
mutants. Phosphorylation of the proteins was performed with the
Ca2+-calmodulin activated MLCK in the solution containing
50 µM protein (in 20 mM imidazole, 50 mM NaCl, 0.1 mM PMSF, pH 7.5), 0.5 µM MLCK, 5.0 µM calmodulin, 0.1 mM CaCl2, 12 mM MgCl2,
and 5 mM ATP. Phosphorylation reaction was carried on for
~2 h at room temperature or overnight on ice. The E22K mutant was
also incubated with 10 µM MLCK-calmodulin for ~2 h at
room temperature (lane 5). Lanes
1 and 2, HCRLC-WT, before (lane
1) and after (lane 2) MLCK treatment;
lanes 3-5, E22K, before (lane
3) and after (lanes 4 and
5) MLCK treatment; lanes 6 and
7, F18L, before (lane 6) and after
(lane 7) MLCK treatment; lanes
8 and 9, R58Q, before (lane
8) and after (lane 9) MLCK treatment.
Note the slower migration of the nonphosphorylated proteins
versus phosphorylated, and the slower migration of the E22K
mutant (pI = 5.10) compared with the HCRLC-WT (pI = 4.92).
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Secondary Structure of HCRLC and Its FHC Mutants--
Far-UV CD
spectroscopy was used to detect changes in the secondary structure of
the HCRLC-WT and its FHC mutants in response to phosphorylation and
Ca2+ binding. The CD spectra for nonmetal bound (apo)
proteins are presented in Fig.
5A. The effect of the FHC
mutation on the HCRLC-WT secondary structure was quantified from
changes in the mean residue ellipticity at 222 nm. This wavelength has
been shown to be sensitive to changes in the
-helical content of the
protein (31, 33). Table II presents the
calculated values (%) of the
-helical content in the apo- and the
Ca2+-bound states as well as the effect of phosphorylation
on HCRLC-WT and its FHC mutants. Data are the average of 2-4
experiments (n), each consisting of 10 scans. The variation
in the
-helical content of the HCRLC-WT and its FHC mutants was
below 2% for the individual experiments. As shown in Fig.
5A and Table II, the
-helical content of HCRLC-WT and its
FHC mutants ranged from 18% to 29%, with the highest for A13T.
Replacement of the alanine residue with threonine (A13T) increased the
-helical content of HCRLC from 18% (n = 4) to 29%
(n = 2). This significant effect of the FHC mutation on
the secondary structure of the HCRLC (p < 0.01) was
quite surprising since alanine has a high potential to form
-helix.
The second significant change was brought about by the E22K mutation,
which resulted in an increase in the
-helical content of HCRLC from 18% to 24% (n = 2, p < 0.01). All
other FHC mutations of HCRLC did not alter its
-helical content in
the apo-state (Fig. 5A and Table II).

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Fig. 5.
Effect of the FHC mutations
(A), Ca2+ binding (B),
and phosphorylation (C) on the CD spectra of HCRLC-WT
and its FHC mutants. Far-UV CD was performed utilizing a 1-mm path
quartz cell in a Jasco J-720 spectropolarimeter. Spectra were
recorded at 195-250 nm with a bandwidth of 1 nm. Mean residue ellipticity
([ ]MRE, in degrees·cm2/dmol) for spectra
was calculated using the following equation: [ ]MRE = [ ]/(10·Cr·l), where [ ] is the
measured ellipticity in millidegrees, Cr is the mean residue
molar concentration, and l is the path length in cm.
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Table II
Effect of the FHC mutations, Ca2+ binding, and
phosphorylation (+ P) on the -helical content of human cardiac RLC
The -helical content for each protein was calculated using the
standard equation for [ ] at 222 nm (31). See "Materials and
Methods." Data are the average of n = 2-4
experiments; each consisting of 10 scans. The variation in the
-helical content was below 2% between independent experiments.
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Effect of Ca2+ Binding--
Fig. 5B and
Table II show the effect of Ca2+ binding on the far-UV CD
spectrum of HCRLC-WT. Similar to other EF-hand Ca2+-binding
proteins, the binding of Ca2+ to HCRLC-WT increased its
-helical content from 18 to 23%. Mean residue ellipticity at 222 nm
for HCRLC-WT (
7681) was similar to previous work by Huang et
al. (30) (
7060) and Wu and Yang (33) (
7500) for rabbit
skeletal muscle RLC. Using the equation utilized in this study to
calculate the
-helical content, these values of the mean residue
ellipticity at 222 nm from Huang et al. (30) and Wu and Yang
(33) would yield 15.6% and 17%, respectively compared with our 18%
for HCRLC-WT (Table II). Similar to HCRLC-WT, the P95A mutant also had
an increase in
-helical content upon Ca2+ binding (Table
II). Interestingly, the binding of Ca2+ to the A13T mutant,
which had the highest
-helical content among all FHC mutants in the
apo-state, caused a decrease (not increase) in its
-helical content
from 29% to 25% (Table II, n = 2, p < 0.05). However, the amount of
-helical content of the
Ca2+-bound A13T was the same as for the other
Ca2+-bound mutants. The binding of Ca2+ to the
F18L mutant produced very little change in its
-helical content
(Table II, n = 3, p > 0.1). The R58Q
mutant did not bind Ca2+ in its nonphosphorylated form, and
Ca2+ did not significantly affect its
-helical content.
However, the phosphorylation restored its Ca2+ binding and
the amount of
-helical content greatly increased on binding of
Ca2+ to phosphorylated R58Q (28%, Table II,
n = 2).
Effect of Phosphorylation--
Phosphorylation of HCRLC-WT did not
change its
-helical content or the binding of Ca2+ to
the phosphorylated form (Table II, n = 4). Only
Ca2+ binding to nonphosphorylated HCRLC-WT had increased
-helical content (23%, Table II, Fig. 5B). No change in
-helix was observed for the phosphorylated F18L and R58Q mutations
in the apo-state. However, a dramatic effect of phosphorylation was
observed for the A13T mutant, whose
-helical content was decreased
from 29% to 19% (n = 2, p < 0.01, Table II, Fig. 5C). The phosphorylated A13T mutant had the
same
-helical content as HCRLC-WT in both phosphorylated and
nonphosphorylated states (Table II). Interestingly, the effect
introduced by the FHC mutation (replacement of the alanine with
threonine) was reversed by the phosphorylation of the mutant (decrease
in
-helix from 29% to 19%). As mentioned above, the R58Q mutation
had the same
-helical content before and after phosphorylation;
however, Ca2+ binding only occurred with the phosphorylated
form and this increased its
-helical content from 20% to 28%
(n = 2, p < 0.01, Table II).
 |
DISCUSSION |
This study investigates the effects of FHC mutations in myosin RLC
on Ca2+ binding, phosphorylation, and secondary structural properties.
Since the ventricular myosin RLC belongs to the superfamily of EF-hand
Ca2+ binding proteins, it was of interest to investigate
the effect of the FHC mutations on its Ca2+ binding
properties. Unlike other EF-hands, RLC contains only one
Ca2+ binding EF-hand domain, between amino acids 37 and 48. We have studied all known FHC RLC mutants with special attention to two mutations, E22K and R58Q, located in the immediate extension of the
helices flanking the HCRLC Ca2+ binding site. Indeed,
whereas A13T, F18L, and P95A mutants decreased the Ca2+
affinity of RLC by 3-fold, the E22K and R58Q mutants had even greater
reduction in Ca2+ affinity compared with HCRLC-WT. The
KCa value for E22K was decreased by 17-fold, and
the R58Q mutant did not bind Ca2+ at all. Interestingly,
Ca2+ binding to the R58Q mutant was restored upon
phosphorylation while the E22K mutant could not be phosphorylated. Even
a 20-fold increase of the MLCK-calmodulin concentration did not result
in phosphorylation of the E22K mutant (Fig. 4).
The R58Q mutation that had Ca2+ binding completely
eliminated, was quite surprising since other EF hand
Ca2+-binding proteins, e.g. troponin C,
calmodulin, contain the Gln residue (and not Arg) in the equivalent
position in the helix C-terminal of the Ca2+ binding site.
The arginine residue of HCRLC, however, is very conserved across
species and a wide spectrum of other RLCs contains the Arg residue in
this position (Fig. 6). It would be
interesting to determine whether substitution of Arg to Gln in these
RLCs would also result in the inactivation of their Ca2+
binding site in the nonphosphorylated state. Interestingly,
phosphorylation of the R58Q mutant at Ser-15 restored the
Ca2+ binding site (Table I). The mechanism of this
intriguing observation is not quite clear. Perhaps the extra negative
charge from the phosphate group of the HCRLC N terminus changes the
conformation of the Ca2+ binding site itself and/or the
region flanking the Ca2+ binding loop, containing the R58Q
residue. It is also possible that these two important regions of RLC,
the Ca2+ binding and the phosphorylation sites, are
communicating with each other in an allosteric manner. This was also
observed for the E22K mutation with a greatly reduced Ca2+
affinity that could prevent phosphorylation. It is worth mentioning that the glutamic acid that follows the phosphorylation site of RLC and
precedes its Ca2+ binding site, is also conserved among
species (Fig. 6). Moreover, the substitution of the positively charged
Lys for the acidic Glu residue resulted in a significant increase in
-helical content of the E22K mutant compared with HCRLC-WT (Table
II). All these changes induced by the E22K mutation are most likely
affecting the interaction of the mutated light chain with the heavy
chain of myosin. One could speculate that this mutation, which
eliminates phosphorylation of the protein and reduces its affinity for
Ca2+, alters working cross-bridges during contraction and
may contribute to the development of hypertrophy in the human heart
(34).

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Fig. 6.
Amino acid sequence alignment of various
isoforms of myosin regulatory light chains. Note that the residues
Phe-18, Glu-22, Arg-58, and Pro-95, which are mutated in the human
ventricular HCRLC of the FHC patients, are very conserved across the
species.
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As mentioned above, the three-dimensional representation of the HCRLC
(Fig. 1B) suggests that mutations A13T, F18L, E22K, and P95A
are located in close proximity to the phosphorylation site of HCRLC.
Thus, any alterations affecting the structure and/or sequence near this
site would be expected to influence the phosphorylation properties of
the HCRLC and/or the relationship between phosphorylation and
Ca2+ binding. The first mutation located near the
phosphorylation site (Ser-15), A13T, resulted in 3-fold decrease in the
KCa value, while phosphorylation of this mutant
caused an additional 6-fold increase in Ca2+ affinity. The
KCa value for phosphorylated A13T was 15 times larger than phosphorylated HCRLC-WT. Therefore, the consequences of the
FHC mutation (A13T) were most profound in conditions where the protein
became phosphorylated. Thus, these results clearly suggest a link
between Ca2+ binding and phosphorylation, as we have
reported previously (35). Unpredictably, substitution of the Ala to Thr
residue resulted in a large increase in the
-helical content (Table
II, Fig. 5A). This was quite surprising since alanine is
known to be predisposed to form
-helical structures. However, this
residue (Ala-13) is located in the region of the RLC, which was not
resolved in the crystal structure of chicken skeletal muscle myosin S1
(1) and it is hard to predict the precise effect of the FHC mutation on
the secondary structure of HCRLC. The
-helical content of the A13T
mutant returned to a normal level (that of HCRLC-WT) upon
phosphorylation. Therefore, phosphorylation of A13T attenuated whatever
sterical constraints were introduced by this FHC mutation. This result
suggests that phosphorylation of HCRLC during contraction could act as
a backup mechanism attenuating the physiological consequences of the
FHC mutation in the working heart. This was also true with the R58Q
mutation whose Ca2+ binding properties were restored upon
phosphorylation. Even in HCRLC-WT, phosphorylation reduced the
Ca2+ dependent elevated
-helical content (Table II).
Our results as well as others (36-39) suggest that phosphorylation of
the regulatory light chains of myosin could have an important physiological role in the regulation of cardiac muscle contraction. Moreover, the relationship between phosphorylation and Ca2+
binding to RLC plays a key role in the working heart (35, 39). One
could speculate that both of these processes may operate as adaptive
and/or protective mechanisms to either attenuate the effect of the FHC
mutations and/or improve performance of the working muscle. Alterations
introduced by the FHC mutations most likely interfere with the
interaction of the RLC with the heavy chain of myosin (40) and affect
the function of myosin cross-bridges during force generation (41). The
region of the myosin heavy chain that contains the RLC has been
postulated to undergo conformational changes that are important for
working muscle (42, 43). The motions of this region of the myosin head
were predicted by crystallographic models (42, 44) and studied further
by fluorescence polarization spectroscopy (45). It was demonstrated
that, during active contraction, the RLC binding domain of the myosin
head undergoes repetitive conformational changes (tilt and twist) and
therefore may play an active role during force generation in muscle.
Therefore, alterations introduced by the FHC mutations could interfere
with the physiological function of the RLC and contribute to
malfunctioning of the human heart. Patients with the FHC RLC mutations
have developed a phenotype of hypertrophic cardiomyopathy, without
sudden death (11, 12). Our results suggest that phosphorylation and
Ca2+ binding to HCRLC may simultaneously act to protect and
attenuate the negative physiological consequences of the FHC mutations. Further work, in progress in our laboratory, is aimed at determining the physiological consequences of these mutations.
 |
FOOTNOTES |
*
This work was supported by American Heart Association Grant
9808237V (to D. S.), National Institutes of Health (NIH) Grant AR45183
(to J. D. P.), and NIH Grants HL06296 and HL26043 (to J. T. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted
to the Swiss Protein Database under Swiss-Prot accesion no. Q14908.
§
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Pharmacology, University of Miami School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33136. Tel.: 305-243-2908; Fax: 305-243-4555;
E-mail: dszczesna@med.miami.edu.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M009823200
 |
ABBREVIATIONS |
The abbreviations used are:
RLC, regulatory
light chain;
HCRLC, human cardiac regulatory light chain;
FHC, familial
hypertrophic cardiomyopathy;
WT, wild-type;
apo, nonmetal bound state;
MLCK, myosin light chain kinase;
PAGE, polyacrylamide gel
electrophoresis;
PMSF, phenylmethylsulfonyl fluoride.
 |
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