(Received for publication, October 11, 1994; and in revised form, December 12, 1994)
From the
Calcium binding proteins mediate a large number of cellular
processes. These processes respond to micromolar fluctuations of
cytosolic calcium in the presence of a large excess of magnesium. The
metal binding sites present in these proteins are either
calcium-specific (regulatory sites) or capable of binding both calcium
and magnesium (structural sites). Using site-directed mutagenesis we
were able to convert the single Ca/Mg
site present in chicken smooth muscle myosin regulatory light
chain (RLC) into a Ca
-specific site. The replacement
of the aspartic acid present in the 12th position (-Z coordinating position) of the metal binding loop with a glutamic
acid increases calcium affinity and abolishes magnesium binding,
rendering the site calcium-specific. To explain this observation, we
hypothesize that restrictions on the ability of side chains to change
conformation, contributing one (for Mg
binding) or
two (for Ca
binding) coordinations could alter the
metal specificity in EF-hands. Other mutations which decrease or
abolish calcium binding have also been characterized. When used to
substitute the endogenous scallop myosin RLC, these mutants were
capable of restoring the Ca
regulation to the
actin-activated myosin ATPase demonstrating that in these hybrid
myosins, the regulatory function of the Ca
-specific
site (present on the essential light chain) does not depend on the
occupancy of the Ca
/Mg
site
(present on the regulatory light chain).
During muscle activation, intracellular calcium levels fluctuate
between 0.1 and 10 µM, while magnesium concentrations are
kept constant at much higher levels (2 mM). Regulatory
proteins must therefore be capable of detecting these changes in
calcium concentrations in the presence of a thousand-fold molar excess
of magnesium. Here we investigate the molecular basis for this
discrimination. The EF-hand Ca
binding motif (1, 2, 3) is responsible for this
discrimination in most regulatory proteins. It consists of a 12-amino
acid binding loop flanked by two perpendicular
-helices (Fig. 1)(1) . Four functional copies of this motif are
found in calmodulin and troponin-C, two in the parvalbumins and one in
the myosin light chains(3, 4) . EF-hands are either
calcium-specific (Ca
sites) or capable of binding
calcium and magnesium (Ca
/Mg
sites). Due to the high concentration of Mg
in
the cytoplasm, Ca
/Mg
sites are
thought to be occupied by Mg
in vivo and
involved in stabilizing the structure of proteins while the
Ca
-specific sites perform the regulatory functions.
The structural basis for divalent metal discrimination by these sites
remains unknown despite the fact that the crystallographic structures
of a number of EF-hand-containing proteins have been
determined(3, 4, 5, 6) . Comparative
studies suggest that Ca
/Mg
discrimination by EF-hand motifs relies on the radius differences
between the ions (1.06 Å for Ca
and 0.76
Å for Mg
) and the consequent energy
requirements for the replacement of the hydration sphere with protein
ligands(7, 8) .
Figure 1:
A, the
structure of an EF-hand calcium binding site showing the coordination
of the metal ion. The EF-hand calcium binding motif is composed of a
loop flanked by two helices. The helix to the NH-terminal
side of the binding loop is shown coming up, perpendicular to the plane
of the picture. The loop containing the coordinating side chains
encircles the calcium ion (pink) in the plane of the picture
and the helix which emerges from the COOH-terminal side of the loop
exits to the right. The oxygens that coordinate the ion directly are
shown in red, the oxygen from the water molecule in white, and the oxygen from the side chain that coordinates the
water molecule in yellow. The first coordinating side chain (X; position 1 in the loop) is hidden under the calcium ion,
below the plane of the figure. The second (Y; position 3) and
third (Z; position 5) coordinating side chains can be observed
as the main chain goes around the calcium ion. The fourth
(-Y; position 7) coordination is provided by a main
chain oxygen and is hidden behind the metal ion. The fifth
(-X; position 9) coordination (yellow) is
indirect and occurs by way of a water molecule (white). The
sixth and seventh (-Z; position 12) are provided by a
bidentate glutamate which reaches in from the first turn of the
COOH-terminal helix and contributes two oxygens. The five coordinating
oxygens from positions 3, 5, 7, and 12 form a pentagon in the plane of
the figure while the oxygens from position 1 and from the water
molecule form the vertices of the two pyramids originating from the
pentagon. The figure was constructed with the coordinates from site III
of troponin-C (19) (5TNC, Brookhaven Data Bank). B,
location of the mutants in the sequence of the binding loop. The
sequence of the cation binding loop of site III of troponin-C (shown
above) and that of site I of smooth muscle RLC are aligned. Also shown
are the metal ion coordination positions (X, Y, Z etc.) and the numbering used for the residues along the loop. The
mutations constructed are shown below the smooth muscle RLC
sequence.
The chicken smooth muscle myosin
regulatory light chain (RLC) (9) has a combination of
characteristics which would be desirable in a model system in which we
can investigate the determinants of ion specificity. It contains a
single Ca/Mg
site which retains
high affinity for metals when removed from myosin, a property not found
in the skeletal or scallop RLCs(10, 11, 12) .
It restores calcium regulation to desensitized scallop myosin, allowing
us to analyze the effects of the mutations on the regulation of the
actomyosin ATPase activity(13) . And finally, although the
crystallographic structure of the smooth muscle RLC (
)has
not been determined, that of skeletal muscle (14) and the
scallop muscle (4) have been elucidated.
Since the crystal
structure of the smooth muscle RLC is not known, we compared EF-hand
calcium binding loops from proteins with known crystal structures and
ion specificity in order to select the positions along the loop most
likely to be involved in Ca/Mg
discrimination(3, 15, 16) . The 4th
position in the loop is occupied by a glycine residue in over 50% of
the known loops and in 100% of the Ca
-specific loops,
suggesting that the extra flexibility and/or small side chain provided
by this glycine might be important for cation discrimination. In the
RLC, this position is occupied by an arginine. The bound cation can
have all its water molecules substituted with protein ligands, as in
the case of the Ca
/Mg
site of
parvalbumin (17, 18) , or keep a single water
molecule, as in the case of the Ca
/Mg
sites of troponin C (19) and the
Ca
-specific sites of calmodulin(20) . It is
the amino acid side chain at position 9 of the calcium binding loop
which determines if a water molecule remains associated with the bound
cation (Fig. 1). If a long side chain is present, for example
glutamic acid in parvalbumin, it coordinates the ion directly. Shorter
side chains, for example serine or aspartic acid, allow for the
presence of a water molecule that is sometimes hydrogen-bonded to the
side chain. The nature of the residue in this 9th position may
influence the size of the coordination sphere and therefore be involved
in ion discrimination. It has also been recently demonstrated that the
residue present in this position determines the ``on'' and
``off'' rates of metal binding to the site(21) . In
oncomodulin it has been demonstrated that the substitution of a
glutamic acid for an aspartic acid in this position has little effect
on the specificity of this site(22) . The 12th position
(-Z coordination) is occupied by a glutamic acid (85%)
or aspartic acid (10%) in most loops irrespective of ion specificity (5, 6, 15, 16, 23) (Fig. 1).
It has been observed in the structure of the sarcoplasmic
Ca
binding protein that the loop containing an
aspartic acid at the 12th position is smaller and more compact when
compared with loops containing glutamic acid at the same
position(5) . Strynadka and James (3) proposed that the
acidic residue at position 12 plays a central role in adapting the loop
of Ca
/Mg
sites when different
cations are bound. The two oxygens from its carboxylate group could
either coordinate the Ca
in a bidentate manner or
change its conformation, providing a single coordination for
Mg
. This conformational change has been observed in
the structure of pike parvalbumin crystallized with either
Ca
or Mg
in its second
site(18) . As RLC possess an aspartic acid at position 12, the
site probably resembles the structure of sarcoplasmic Ca
binding protein and we would expect that the substitution of
aspartic acid for glutamic acid could open the site and favor the
binding of larger ions.
Site-directed mutants were constructed to tests these hypotheses. We found that the replacement of the aspartic acid present in the 12th position with a glutamic acid renders the site calcium-specific. Other mutants increased, decreased, or abolished calcium affinity without drastically altering the specificity. All mutants were used to replace the endogenous RLC from scallop muscle. Since the mutants restored calcium regulation and the calcium-specific site present in the essential light chain, we conclude that in these hybrids the occupancy of the RLC site is not essential for proper regulation of the myosin ATPase.
The numbers refer to the position of the
mutated amino acid in the EF-hand site. The double mutant R4G/D12E was
generated by performing the second mutation D12E on the R4G mutant
template. The mutated coding region of the gene was sequenced and used
to replace the wild type fragment of the cDNA between the single HindIII/EcoRI sites in pMW172(25) . The
presence of the mutations was confirmed by sequencing the final
expression vector. Expression of smooth RLC was performed as
described(25) . The bacterial pellet was resuspended in 25%
sucrose, 2 mM EDTA, 2 mM EGTA, 50 mM Tris-Cl, pH 8.0, and lysed in a French Press (16,000 p.s.i.).
After lysis, trichloroacetic acid was added to a final concentration of
3%, and the precipitated protein was resuspended in 25 mM Tris-Cl, pH 7.5, 0.5 mM MgCl, 2 mM
-mercaptoethanol. The pH was adjusted to 7.0 with Tris base,
and the suspension was dialyzed overnight against the same buffer.
After centrifugation, 6 M urea was added to the supernatant.
The pellet was resuspended in 6 M urea in the same buffer.
The solutions were clarified by centrifugation and separately
applied to a DEAE-Sepharose FF equilibrated with 6 M urea, 25
mM Tris-Cl, pH 7.5, 0.5 mM MgCl, 2 mM
-mercaptoethanol. The pure proteins were eluted with a
0-400 mM NaCl gradient. The final purification was
achieved on Phenyl-Sepharose 6B equilibrated in 4 M NaCl, 25
mM Tris-Cl, pH 7.5, 0.5 mM MgCl
using a
gradient from 4.0 to 0 M NaCl.
where y is the number of sites filled at each
[Ca] and K
is the apparent dissociation
constant. Since saturation occurred at values higher than 1 mol of
Ca
/mol of protein, a correction factor (n`)
was determined during the fitting procedure to account for nonspecific
binding. Nonspecific Ca
binding to the surface of the
dialysis membrane and the vessel can be demonstrated in the absence of
protein. The ability of magnesium to compete off the bound calcium was
determined as follows. RLC was added to the upper chamber at the same
concentration used in the Ca
measurements, in the
same buffer. At the tenth fraction, the calcium concentration was
increased by 40 µM, and aliquots of 2 to 40 µl of 250
mM MgCl
were added every fifth fraction, resulting
in a final concentration of 10 mM after the ninth addition. At
the end of the titration, CaCl
was added to a final
concentration of 10 mM. The fourth and fifth fractions after
each addition were used to determine the amount of calcium bound. K
was determined by fitting
three data sets to , using a nonlinear regression
curve-fitting procedure.
where y` is the number of sites filled at each
[Mg] concentration, K
and K
are the apparent dissociation
constants, and n` a correction factor to nonspecific binding. K
was obtained from .
As expected for a single
Ca/Mg
binding site, wild type RLC
binds approximately 1 mol of Ca
per mol of RLC (Fig. 2A). In the presence of magnesium, this ability
is almost abolished (Fig. 2B). The substitution of the
arginine in position 4 of the loop with a glycine (R4G, Fig. 1)
was a silent mutation. As the wild type RLC, it binds calcium with an
affinity close to 10 µM (Fig. 2A; Table 1), and its affinity for calcium is reduced 9-fold (to 90
µM) in the presence of 2 mM Mg
(Fig. 2B; Table 1). The affinity of wild
type RLC and mutant R4G for magnesium is close to 400 µM (Fig. 2C; Table 1).
Figure 2:
Calcium
binding to isolated recombinant smooth muscle RLC () and mutants:
D9S (
), D12A (
), R4G (
), D12E (
), D9E
(
), and R4G/D12E (
).
Ca
binding was measured as a function of Ca
concentration in the absence (A) and presence (B) of 2 mM Mg
. In C, the
capacity of Mg
to displace bound
Ca
was measured as a function of
Mg
concentration. The data points represent the mean
from three independent assays. Standard deviations of the mean are
shown as bars (n = 3). The lines are
curve-fitted according to the parameters shown in Table 1.
In the second set of
mutants, we changed the length of the side chain in position 9,
replacing the aspartic acid by either a glutamic or a serine residue
(D9E and D9S, Fig. 1). When compared with the wild type RLC,
these mutants showed a 2-fold reduction in calcium affinity both in the
presence and absence of magnesium (Fig. 2, A and B; Table 1) and also a reduction in magnesium affinity (Table 1). This decrease in affinity did not result in a
significant change in ion selectivity as defined by the ratio K/K
(Table 1).
A
dramatic increase in ion selectivity was obtained when the side chain
of position 12 was made longer by replacing the aspartic with a
glutamic acid (D12E and R4G/D12E, Fig. 1). When compared with
wild type RLC, we observed in both mutants a 2-fold increase in
affinity for calcium in the absence of magnesium (Fig. 2A) and a 100-fold increase in affinity in the
presence of 2 mM Mg (Fig. 2B; Table 1). This rendered the site
calcium-specific, and bound calcium could not be displaced by up to 10
mM magnesium (Fig. 2C). The lower saturation
values observed for mutants D12E and D12E/R4G in the presence of
Mg
(Fig. 2, A and B) are
probably related to a reduction in nonspecific Ca
binding (Table 1).
These results demonstrate that the
Ca/Mg
binding site present in the
isolated protein was transformed into a Ca
-specific
site.
Figure 3: Reconstitution of desensitized scallop myofibrils with recombinant and smooth RLCs. A, SDS-polyacrylamide gel electrophoresis (15%); B, urea-glycerol gel of desensitized scallop myofibril (DMF) reconstituted with the following regulatory light chain: 1, none; 2, smooth wild type; 3, smooth D12A; 4, scallop RLC; 5, smooth D9S; 6, smooth R4G; 7, smooth D12E; 8, smooth D9E; 9, smooth R4G/D12E. The arrows indicate the regulatory light chain. Note that in SDS gels the scallop RLC co-migrates with the ELC.
Figure 4:
Ca binding properties of
desensitized scallop myosin reconstituted with recombinant smooth RLCs. A,
Ca
binding to scallop myosin
as a function of Ca
concentration. Desensitized
myosin (
) was reconstituted with wild type smooth RLC (
) or
smooth RLC D12A (
). The data points represent the mean from
three independent assays. B, urea/glycerol polyacrylamide gel
electrophoresis of desensitized myosin alone (1) and
reconstituted with wild type smooth muscle RLC (2) and D12A (3). The arrow indicates the
RLC.
The fact that the increase in calcium
selectivity was obtained without a decrease in calcium affinity (Table 1) suggests that the structural modification caused by the
mutation does not affect the Ca binding but
drastically reduces Mg
binding. Since Mg
is smaller than Ca
, we would intuitively expect
that an increase in the length of the side chain in the 12th position
should favor the binding of the smaller ion, contrary to what we
observed. Indeed, our results with the mutants in the 9th coordinating
position where we both increased (D9E) or decreased (D9S) the length of
the side chain without detecting changes in cation specificity support
the idea that the specificity is not determined directly by the length
of the coordinating side chain. In pike parvalbumin, where the crystal
structure of both the Ca
and Mg
forms have been determined(18) , it was found that upon
Mg
binding the major change in the loop to
accommodate the smaller ion was a rotation of the side chain of the
residue in the 12th coordinating position (-X position).
In the presence of Ca
, the two carboxylate oxygens of
this side chain form part of seven coordinating oxygens organized into
a bi-pentagonal pyramid. In the presence of Mg
, this
side chain rotates so that it only contributes one ligand to the
six-membered octahedral coordination shell. This type of rotation has
been postulated as the conformational change necessary to accommodate
the smaller Mg
ion(3) .
We would like to
propose that restrictions on the rotation of coordinating side chains
contribute to the determination of the ion specificity of EF-hands.
Accordingly, in the wild type smooth muscle RLC, the aspartate side
chain in the 12th position could rotate and accommodate either
Mg or Ca
. In the mutant protein,
this side chain would have its rotation sterically blocked into the
bidentate configuration. The site would not be able to accommodate the
smaller Mg
ion, thereby reducing its affinity for
this ion. Furthermore, if the longer glutamic side chain is locked in
the bidentate orientation, preferred by the Ca
ion,
and cannot rotate to provide the single coordination necessary to
accommodate the Mg
ion, it is expected that the
affinity for Mg
would be greatly reduced. This
restriction in rotation should also favor calcium binding, increasing
the affinity for this ion, as we observed in the D12E mutant. Rotation
of side chains, although predicted (3) and observed
before(18) , have not been postulated as a possible explanation
for ion selectivity. Direct evaluation of this hypothesis must await
the determination of the structure of the smooth muscle RLC.
This idea could also explain why it has been impossible to predict the ion specificity of EF-hands based only in the primary sequence. Because the rotation is limited by the local environment of the side chain, it would be difficult, if not impossible, to predict from the primary structure if a given side chain is able to rotate in a particular environment.
When used to replace
the endogenous RLC in scallop myosin, our mutant smooth muscle RLCs
were capable of restoring calcium regulation to the system. The
observation that the calcium-specific mutant (D12E) was able to inhibit
in the absence of calcium (a situation where the site should be empty)
suggested that the occupancy of the RLC site is not necessary for
inhibition. This difference in behavior when compared with the results
obtained with the skeletal or scallop system(11, 12) was confirmed when we tested a smooth muscle RLC with the
Ca/Mg
site destroyed (D12A). To
exclude the possibility that the D12A mutant of smooth muscle RLC
regained its metal binding site when incorporated into the scallop
myosin, we measured calcium binding to the reconstituted myosins. These
measurements demonstrated that this mutant is capable of restoring the
calcium-specific, regulatory site as expected from its ability to
restore the regulation of the ATPase.
These results demonstrate that
it is possible to obtain hybrids between scallop myosin and smooth
muscle RLCs with a single Ca site still capable of
being regulated by calcium. This strongly supports the idea that the
RLC Ca
/Mg
is not necessary for the
reconstitution of the Ca
-specific site on the ELC or
for the regulation of the ATPase. Rather, the RLC
Ca
/Mg
site probably plays a
structural role, as expected from its specificity. It remains to be
determined why it has not been possible to obtain similar results with
hybrids constructed using mutants of the endogenous or skeletal muscle
RLCs.