(Received for publication, October 31, 1994; and in revised form, December 1, 1994)
From the
Neurocalcins are brain-specific proteins that belong to a new
subclass of the EF-hand superfamily of calcium binding proteins,
defined by the photoreceptor cell-specific protein, recoverin.
Recoverin, which regulates the desensitization of photo-excited
rhodopsin, is myristoylated and exhibits a calcium-myristoyl switch.
Like recoverin, neurocalcins have a signal for N-myristoylation and possess four EF-hands, although the first
one lacks some residues critical for calcium binding. In this work, I
have examined the calcium and membrane binding properties of
recombinant myristoylated and unmyristoylated neurocalcin . I show
that neurocalcin, like recoverin, binds to biological membranes in a
calcium- and myristoyl-dependent manner. Both myristoylated and
unmyristoylated proteins bind three calcium ions. However, the
unmyristoylated form exhibits a higher affinity for calcium than the
myristoylated protein but shows a lower cooperativity in binding
calcium. These data support the model for the calcium-myristoyl switch
mechanism proposed for recoverin (Zozulya, S., and Stryer, L.(1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11569-11573; Dizhoor,
A. M., Chen, C. K., Olshevskaya, E., Sinelnikova, V. V., and Hurley, J.
B. (1993) Science 259, 829-832). Using point mutations,
I have investigated the relative importance of each of the three
functional EF hands (EF2, EF3, and EF4) in the calcium and membrane
binding properties of neurocalcin. Calcium and membrane binding
properties of the mutant-myristoylated proteins suggest that binding of
calcium to EF2 is critical in triggering the binding of the protein to
membranes.
In recent years, a new branch of the EF-hand superfamily of
calcium binding proteins (1, 2, 3) has
emerged. It comprises several related molecules that are expressed
mainly in the brain or in photoreceptor cells of various vertebrates.
The most extensively studied members of this new subfamily, the bovine
recoverin and the frog S-modulin, are essential Ca sensors in rod photoreceptor cells, which regulate in a
Ca
-dependent manner the desensitization of the
light-activated
rhodopsin(4, 5, 6, 7, 8) .
Similar photoreceptor-specific proteins have been characterized in
chicken, mice, and human species(9, 10, 11) .
X-ray studies of the three-dimensional structure of the
Ca-bound form of recoverin has revealed a new folding
pattern consisting of two pairs of EF-hands that are closely packed
together(12, 13) . The amino terminus of native bovine
retinal recoverin is heterogenously acylated by a small family of fatty
acids, the most abundant being myristoleate(14) . Subsequent
studies have shown that myristoylated recoverin binds to membranes in
the presence of Ca
and that this binding requires the
amino-terminally attached myristoyl group(15, 16) .
Several groups have reported the purification and/or molecular
cloning of cDNAs encoding proteins homologous to recoverin that are
specifically expressed in the nervous system of various vertebrates. In
rat brain, four different proteins, called NVP1, NVP2, NVP3, and
hippocalcin, have been characterized from their cDNA
sequence(17, 18, 19) , each exhibiting a
specific spatial pattern of expression in the brain. Several isoforms
of the so-called neurocalcin were purified from bovine
brain(20) . The cDNA sequence of a particular isoform,
neurocalcin , as well as peptide sequences of several other
isoforms revealed that these molecules are highly similar to the rat
proteins as well as to a chicken developmentally regulated protein,
GGVILIP(20, 21, 22) . Another member of this
subfamily was recently identified in Drosophila(23) .
The protein, called frequenin, was shown to be overexpressed in a Drosophila mutant that exhibits a frequency-dependent increase
in synaptic facilitation at the neuromuscular junction. These findings
suggest that the vertebrate brain-specific homologs of recoverin might
have a similar physiological role of regulating the synaptic plasticity
in a calcium-dependent manner. However, no function has been assigned
yet for any of them. All of these proteins exhibit more than 45%
identity with recoverin and share the following conserved features: (i)
a 190-200 amino acid chain that contains a consensus
myristoylation signal at the amino terminus; (ii) two or three
classical EF-hand signatures (1, 2) (EF2, EF3, and
EF4); and (iii) a sequence, EF1, which has a poor matching score with
the consensus EF-hand signature and contains a conserved Cys-Pro pair.
In the x-ray structure of recoverin, however, this sequence exhibits a
helix-loop-helix folding pattern like that of a classical EF-hand,
although it lacks several amino acids required for Ca
binding(12) . These conserved characteristics suggest
that all of the members of this subfamily possess, like recoverin, a
Ca
-myristoyl switch that is likely to be important in
Ca
signaling. Indeed, this has been shown for the rat
brain hippocalcin isoform(24) .
To investigate the mechanism
of the calcium-myristoyl switch, I have characterized the calcium and
membrane binding properties of both myristoylated and unmyristoylated
recombinant neurocalcin . I have shown that myristoylation of
neurocalcin decreases its affinity for calcium but increases the
cooperativity of calcium binding. These results support the original
model for the calcium-myristoyl switch
mechanism(15, 16) , which postulates that, upon
binding of calcium, the myristoyl group is extruded from a hydrophobic
pocket of the protein and becomes available to insert into biological
membranes. The role of individual EF-hands in the calcium-myristoyl
switch were further dissected by constructing and characterizing
mutants of neurocalcin disabled in each of the three functional
EF-hands. Calcium and membrane binding properties of the mutant
myristoylated proteins suggest that binding of calcium to EF2 is
critical in triggering the binding of the protein to membranes.
Recombinant myristoylated or
unmyristoylated neurocalcin were purified from the corresponding
bacterial extract by a two-step procedure consisting of
Ca
-dependent hydrophobic chromatography on
phenyl-Sepharose followed by ion-exchange chromatography on Q-Sepharose
as previously described(25) . About 10-20 mg of purified
protein were typically obtained from 1 liter of culture. The
neurocalcin content in purified preparations was determined from the
absorbance at 280 nm using a molar extinction coefficient (calculated
from the amino acid content of the molecule using values of 5.5 and 1.4
for
of tryptophan and tyrosine,
respectively) of 20 mM
cm
.
The tritium-labeled myristoylated
neurocalcin was purified similarly from a 200-ml culture of
DH5
/pBB131/pDL1312 in LB medium supplemented with 100 µg/ml
ampicillin, 100 µg/ml kanamycin, and 10 µCi/ml
[
H]myristic acid (39.3 Ci/mmol) (DuPont NEN). The
specific radioactivity of the purified labeled neurocalcin was 5
10
cpm/nmol of protein.
For the
binding assays, 1-ml samples of a 30-50 µM solution
of calcium-free neurocalcin in 25 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 0.2 mM MgCl were placed in the
top compartment of Centricon-10 concentrators (molecular mass cut-off
of 10 kDa). 20 µl of a 500-µM CaCl
solution containing about 10
cpm of
CaCl
were added to the protein and thoroughly
mixed by vortexing. 20 µl of this solution were taken for counting
of total radioactivity in a scintillation counter. The mixtures were
then centrifuged at room temperature in a table-top I.E.C. centrifuge
for 15-20 s until 25-30 µl were filtrated through. The
ultrafiltrate was added back to the protein solution, which was again
mixed and centrifuged as above (this second centrifugation was done to
alleviate a filtration membrane dead volume of about 10 µl).
Radioactivity of a 20-µl sample of the filtrate, which contained
the free calcium, was determined by scintillation counting. Then, 20
µl of a 500-µM unlabeled CaCl
solution
were added to the top compartment, and the centrifugation procedure
described above was repeated. Successive additions of unlabeled calcium
were done to cover the range of desired total calcium concentrations
(up to 500 µM). After each addition of cold ligand, the
ratio of radioactivity in 20 µl of filtrate/radioactivity in 20
µl of top compartment (initial measurement) was equal to free
calcium/total added calcium. This allowed me to calculate the number of
bound calcium ions per neurocalcin molecule as a function of free
calcium.
For the binding assays, 10 µg of purified
neurocalcins were incubated with brain membranes (5 µg of proteins)
in 60 µl of 50 mM MOPS, pH 7.1, 0.1 M KCl
containing either 1 mM EGTA or 1 mM EGTA and 1.05
mM CaCl (free Ca
> 50
µM). After 15 min of incubation at 22 °C, the mixtures
were centrifuged for 10 min at 30 p.s.i. (100,000
g)
in an airfuge (Beckman) at room temperature. Both supernatant and
pellet fractions were denatured in an equal volume of sample buffer,
and identical volumes were electrophoresed on a 15% SDS
gel(28) , which was stained with Coomassie Blue. Binding of
H-myristoylated neurocalcin
(3 µg) to brain
membranes was measured similarly except that a set of Ca-EGTA buffers,
prepared as described(29) , were used to control the free
Ca
concentrations from 200 nM to 5
µM. The free Ca
concentration of these
buffers was determined by using the fluorescent dye indicator rhod-2
(Molecular Probes). To set calcium concentrations above 10
µM, known amounts of total calcium were added to the
protein solutions in calcium-free buffers. The concentration of free
calcium was assumed to be equal to total calcium added minus the
concentration of calcium binding site provided by the protein (which
was small in comparison to total calcium added). After high speed
centrifugation in airfuge, the supernatants and pellets, resuspended in
60 µl of 50 mM Tris-HCl, pH 8, 1 mM EGTA, were
spotted on Beckman Ready Cap
, and the radioactivity was
measured in a scintillation counter. For any given sample, the fraction
of neurocalcin bound was calculated from the ratio of the cpm in the
pellet to that in the pellet plus supernatant.
The membrane binding
properties of the mutant neurocalcins were investigated by incubating
the proteins (3 µM) with bovine brain membranes at various
calcium concentrations. After centrifugation at 13,000 g for 15 min, the pellet fractions were resolved on
SDS-polyacrylamide gel electrophoresis, and the relative amounts of
neurocalcin in each sample were determined by densitometry of the
Coomassie Blue-stained gels.
Figure 1:
SDS gel analysis of purified
recombinant neurocalcins. Samples of 4 µg of the various purified
protein preparations were electrophoresed on a 13.5% SDS gel and
stained with Coomassie Blue. Lane1, standard
proteins (94, 67, 43, 30, 21.1, and 14.4 kDa, from top tobottom); lane2, unmyristoylated
neurocalcin ; lane3, myristoylated neurocalcin
; lane4, myristoylated ME84Q; lane5, myristoylated-ME120Q; lane6,
myristoylated ME168Q; lane7, standard
proteins.
The stoichiometry of
Ca binding to recombinant neurocalcins was determined
by ultrafiltration using Ca
-free preparations of
protein obtained by chromatography through a
Ca
-chelating column (see ``Materials and
Methods''). As shown in Fig. 2, both unmyristoylated and
myristoylated neurocalcin bound three calcium ions per molecule at
saturating calcium concentrations. The macroscopic association
constants for each of the three calcium binding sites were deduced by
fitting the calcium binding curves to the Adair equation (Table 1). Calcium binding to each site occurred at micromolar or
submicromolar calcium concentrations, indicating that neurocalcin can
respond to physiological changes in the concentration of this
messenger.
Figure 2:
Myristoylation alters the calcium affinity
of neurocalcin . Calcium binding was determined as described under
``Materials and Methods.'' The protein concentration was 40
µM. Unmyristoylated neurocalcin (
) and myristoylated
neurocalcin (
) in presence of 0.2 mM MgCl
and myristoylated neurocalcin in presence of 5 mM MgCl
(
) are shown. The curves show the
computer-derived fits from the Adair modeling using the stoichiometric
constants listed in Table 1. Similar data were obtained on three
independent preparations of myristoylated and unmyristoylated
neurocalcin.
Binding of calcium ions to the myristoylated neurocalcin
was cooperative as indicated by the fact that the association constant K, which characterized the binding of the second
calcium ion to the protein, is much greater than the constant K
characterizing the binding of the first one.
Hence, binding of the first calcium ion to the myristoylated
neurocalcin strongly favors the binding of a second one. Similarly, a
Hill plot of the same data gave a Hill coefficient higher than 2 (not
shown). These data indicate that the transition between the
calcium-free and calcium-bound form is highly concerted. By contrast,
unmyristoylated molecule binds calcium with a higher affinity than the
myristoylated protein but without noticeable cooperativity (Table 1). In the presence of 5 mM MgCl
, the
apparent affinity for calcium of the first two Ca
binding sites of myristoylated neurocalcin
was decreased ( Fig. 2and Table 1), indicating that Mg
was able to weakly compete with Ca
at these
sites.
Figure 3:
Tryptophan emission spectra of
myristoylated and unmyristoylated neurocalcin in presence and
absence of calcium. A, purified proteins (3 µM)
with 100 µM CaCl
or 2 mM EGTA were
excited at 290 nm. Fluorescence emission spectra were recorded from 300
to 400 nm. Background fluorescence was recorded similarly in the
absence of added protein and subtracted from the spectra. B,
calcium dependence of the fluorescence emission intensity at 336 nm of
1 µM protein solutions of myristoylated (
) or
unmyristoylated (
) neurocalcin
was measured as
described under ``Materials and Methods.'' Free calcium
concentrations up to 10 µM were set by
Ca
-EGTA buffers. Above this value, known amounts of
total calcium were added to the protein solutions in calcium-free
buffers.
Figure 4:
Ca and
myristoyl-dependent binding of neurocalcin
to bovine brain
membranes. Myristoylated and unmyristoylated neurocalcin
were incubated with bovine brain membranes either with 1
mM EGTA (-Ca
) or with 1 mM EGTA and 1.05 mM CaCl2 (+Ca
).
After high speed centrifugation, equal volumes of supernatant and
pellet were electrophoresed on 15% SDS gels, which were stained with
Coomassie Blue. Note the Ca
-induced shift in
electrophoretic mobility. Controllanes correspond to
membranes incubated without neurocalcin.
The
Ca dependence of membrane binding of myristoylated
neurocalcin
was determined by using a
H-myristoyl-labeled protein. As shown in Fig. 5,
binding of myristoylated neurocalcin
to membranes was
half-maximal at 0.75 ± 0.1 µM Ca
and cooperative with respect to Ca
(Hill
coefficient = 2.3 ± 0.2; means of five independent
determinations). Again, Mg
was ineffective in
promoting binding of myristoylated neurocalcin
to brain membranes
(not shown). Proteolytic treatment of the membranes only slightly
reduced the maximal amount of binding of the labeled protein and had
little effect on the Ca
dependence (Fig. 5).
Hence, specific membrane protein receptors do not seem to be required
for Ca
-induced binding.
Figure 5:
Ca-dependent binding of
H-myristoylated neurocalcin
to bovine brain
membranes. Membrane binding was determined as described under
``Materials and Methods'' on untreated (
) or
trypsin-treated (
) membranes.
The
Ca-dependent binding of native brain neurocalcins to
membranes was also examined. Crude extract of bovine brain, containing
both cytosolic and membrane components, was centrifuged at high speed
either in the presence or absence of Ca
, and the
supernatant and pellet fractions were analyzed by Western blots using
anti-neurocalcin antibodies. Native bovine brain neurocalcins were
indeed found to be associated with membranes at micromolar calcium
concentrations (data not shown).
As shown in Fig. 6, at 200
µM Ca, each of the three modified
neurocalcins bound only 2 mol of calcium ions/mol of protein. As
expected, the glutamate to glutamine mutation in each of the three
EF-hands abolished the binding of calcium at the corresponding site.
Interestingly, in each case, the impairment of one calcium binding site
profoundly affected the two remaining ones. These results are
consistent with a tight coupling between the three calcium binding
sites in the native protein. Mutation of EF3 was the most detrimental
to calcium binding at the other two sites.
Figure 6:
Calcium binding properties of mutant
myristoylated neurocalcins containing glutamine instead of glutamate in
an EF-hand. Calcium binding was determined as described under
``Materials and Methods.'' The protein concentration was 40
µM except for ME120Q, which was at 20 µM. Symbols used are as follows: , wild type;
, ME84Q
(in EF2);
, ME120Q (in EF3);
, ME168Q (in EF4). Identical
results were obtained in four experiments using two independent
preparations of ME84Q and ME120Q and three experiments on a unique
preparation of ME168Q.
Figure 7:
Tryptophan emission spectra of wild-type
or mutated myristoylated neurocalcins. Purified proteins (3
µM) in 1 mM EGTA (A) or in 500
µM CaCl (B) were excited at 290 nm,
and fluorescence emission spectra were recorded from 300 to 400 nm.
Background fluorescence was recorded similarly in the absence of added
protein and subtracted from all spectra. In A, the
fluorescence spectra were, from top to bottom,
wild-type myristoylated neurocalcin, ME168Q, ME120Q, and
ME84Q.
Figure 8:
Calcium dependence of the fluorescence
intensity of myristoylated wild-type or mutated neurocalcins. The
fluorescence emission intensity at 336 nm of 1 µM protein
solutions was measured as described under ``Materials and
Methods.'' Free calcium concentrations up to 10 µM were set by Ca-EGTA buffers. Above this value,
known amounts of total calcium were added to the protein solutions in
calcium-free buffers. Symbols are the same as in Fig. 6. Similar data were obtained in four experiments using two
independent preparations of ME84Q and ME120Q and in two experiments on
a unique preparation of ME168Q.
Figure 9: Membrane binding properties of mutant neurocalcins. The membrane binding properties of the wild-type and mutant neurocalcins were determined as described under ``Materials and Methods.'' Symbols are the same as in Fig. 6. The errorbars indicate the standard error means of three experiments.
In the present work, I have investigated the calcium and
membrane binding properties of a recombinant bovine brain neurocalcin
expressed in E. coli. Neurocalcin
, either
myristoylated or unmyristoylated, was found to bind three calcium ions
with affinities in micromolar and submicromolar range. This
demonstrated that the three predicted EF-hands of neurocalcin are
functional. Nakano et al.(30) , however, found only
two calcium binding sites for a recombinant unmyristoylated neurocalcin
purified from E. coli. At present, I have no
explanation for this discrepancy. The present findings, however, were
clearly confirmed by results of site-directed mutagenesis of the three
putative EF-hands. Replacement of the critical glutamic acid located in
position 12 of the calcium binding loops by a glutamine in EF2, EF3, or
EF4 eliminated, in each case, one calcium binding site. Hence these
three EF-hands are the functional calcium binding sites of neurocalcin
. EF1, which has a meager matching score with the
consensus EF-hand signature, was only inferred from the crystal
structure of the homologous protein, recoverin(12) . In
crystals of recoverin, EF1 exhibits a nearly classical helix-loop-helix
structure that is paired with EF2. However, EF1 in recoverin, as in all
the other members of the subfamily of recoverin-like proteins, lacks
some of the critical ligands for calcium and contains a Cys-Pro pair in
the loop, which distorts a favorable Ca
binding
geometry(12) . Indeed, no calcium was detected in EF1 in the
crystal structure of recoverin. The present data confirm that, in
solution, this EF-hand in the homologous neurocalcin is also
nonfunctional.
The second observation reported here is the finding
that myristoylation of neurocalcin has a pronounced effect on the
affinity of the molecule for calcium. The unmyristoylated form
exhibited a higher affinity for calcium than the myristoylated protein.
The latter, however, showed a higher cooperativity in binding of
calcium than the former. A similar observation has been made on
recoverin, ()and it is likely that this reflects a key
property of this type of calcium binding proteins that exhibit a
calcium-myristoyl switch. The model for the calcium-myristoyl switch
mechanism (15, 16) postulates that, upon binding of
calcium, the myristoyl group is extruded from a hydrophobic pocket of
recoverin and becomes available for insertion into biological
membranes. In absence of membranes, however, this process should be
disfavored because it exposes a hydrophobic fatty acid chain to the
aqueous environment. In other words, part of the binding energy of
calcium is used for solvation of the myristoyl group. These energetic
constraints do not apply for the unmyristoylated protein, which should
thus be expected to bind calcium with a higher affinity, as observed.
The third point highlighted by this study is the strong interdependence of the three functional EF-hands of neurocalcin in the binding of calcium. This was illustrated first by the high cooperativity in calcium binding of the myristoylated protein and was further confirmed by site-directed mutagenesis of each of the three EF-hands. In each case, the disabling of one EF-hand strongly decreased the affinity for calcium of the other two calcium binding sites. Interestingly, each of the three mutations affected differently the affinity for calcium of the two remaining binding sites (Fig. 7). Mutation of EF3 had the most pronounced effect on calcium binding at the two other sites, while mutation in EF2 had the least effect.
These observations can be rationalized in the light of the three-dimensional structure of the homologous molecule, recoverin. Given the degree of identity/similarity of the primary sequences of neurocalcin and recoverin (45% identity, 15% additional similarity), it is highly probable that these two proteins have a similar folding pattern. Recoverin is a compact protein made of two domains, each containing a pair of EF-hands, EF1/EF2 and EF3/EF4. In addition, EF2 and EF3 are in contact with each other at the interface of the two domains. This particular arrangement can account for why alteration of any EF-hand affects the binding of calcium to the other two. Furthermore, because EF3 contacts both EF2 and EF4, it is not surprising that inactivation of this particular EF-hand has the most pronounced effect on calcium binding by the other two sites.
All of
the known members of the recoverin/neurocalcin subfamily possess a
consensus myristoylation signal and are likely to be myristoylated in vivo as it has been shown for recoverin. Indeed, the amino
terminus of several of these isoproteins purified from the brain is
blocked(20) . I have shown here that the covalent addition of a
myristoyl group at its amino terminus conferred on neurocalcin the
ability to interact in a Ca-dependent manner with
biological membranes. This binding was cooperative with respect to
Ca
and occurred at submicromolar Ca
concentrations, i.e. in the physiological range. The
endogenous neurocalcins obtained from bovine brain exhibited the same
Ca
-dependent association with membranes, which
strongly supports the idea that native brain neurocalcins are
myristoylated. Hence, neurocalcins exhibit in vitro the same
Ca
-myristoyl switch as retinal recoverin and the rat
brain hippocalcin. It is likely that this property will be found as a
hallmark of this subfamily of EF-hand-containing calcium binding
proteins.
Analysis of the membrane binding properties of the mutant myristoylated neurocalcins revealed the critical role of EF-hand 2 in the calcium-myristoyl switch. The calcium dependence for the association of mutant ME84Q with membrane indicated that binding of calcium to the two functional calcium binding sites, EF3 and EF4, was not enough to trigger efficient binding of the protein to membranes. In contrast, for mutant neurocalcins ME120Q and ME168Q, mutated in EF3 and EF4, respectively, the calcium dependence of membrane binding was roughly identical to that of calcium binding. From these data, I propose that calcium occupancy of EF2 is necessary to induce a conformational change that leads to the association of myristoylated neurocalcin with membranes. Why then does ME84Q bind to membranes at high calcium concentrations? One attractive hypothesis is that membranes increase the calcium affinity of the disabled EF2. Indeed, it has been shown that calmodulin binding peptides could restore normal calcium binding to calmodulin EF-hands that were disabled by a Glu-to-Ala mutation in position 12 of the loop(31) . Membranes could play a similar role in the case of myristoylated mutant neurocalcin. The fluorescent properties of ME84Q seem to indicate, indeed, that even in the absence of membrane, a calcium ion can bind to mutated EF2 at concentrations above 1 mM (Fig. 8). Further studies will be required to clarify this issue.