©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium and Membrane Binding Properties of Bovine Neurocalcin Expressed in Escherichia coli(*)

(Received for publication, October 31, 1994; and in revised form, December 1, 1994)

Daniel Ladant(§)

From the Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


MATERIALS AND METHODS

Expression in Escherichia coli and Purification of Unmyristoylated and Myristoylated Recombinant Bovine Neurocalcin

Expression and purification of recombinant bovine neurocalcin has been previously described(25) . Briefly, a bovine neurocalcin cDNA was amplified by polymerase chain reaction and subcloned into plasmid vector pTREC2(13) . In the resulting expression vector pDL1312, the neurocalcin coding region was placed under the transcriptional control of the strong heat-inducible p(r) promoter of phage. E. coli strain DH5alpha harboring pDL1312 was grown in LB medium at 32 °C until mid-log phase, and then synthesis of neurocalcin was induced by increasing the growth temperature to 42 °C. Bacteria were harvested after 2 h of induction. Recombinant myristoylated neurocalcin was obtained by coexpressing neurocalcin and Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase in E. coli. The compatible plasmid pBB131, which carries the myristoyl-CoA:protein N-myristoyltransferase gene under the control of isopropyl-1-thio-beta-D-galactopyranoside-inducible tac promoter(26) , was cotransformed in DH5alpha with pDL1312. The cells were grown in LB at 32 °C until mid-log phase; then, synthesis of myristoyl-CoA:protein N-myristoyltransferase was induced by addition of isopropyl-1-thio-beta-D-galactopyranoside, and, later, the growth temperature was shifted to 42 °C to trigger the synthesis of neurocalcin. To ensure complete myristoylation, 10 µg/ml myristic acid were added to the culture medium.

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 DH5alpha/pBB131/pDL1312 in LB medium supplemented with 100 µg/ml ampicillin, 100 µg/ml kanamycin, and 10 µCi/ml [^3H]myristic acid (39.3 Ci/mmol) (DuPont NEN). The specific radioactivity of the purified labeled neurocalcin was 5 times 10^5 cpm/nmol of protein.

Site-directed Mutagenesis

Site-directed mutagenesis of glutamates 84, 120, or 168 to glutamine were performed by standard polymerase chain reaction reactions using pDL13 as a template and the following oligonucleotide primers: MEQ84 (for Glu-84 to Gln mutation), 5`-TAGACTTTAGACAATTCATCATAG-3`; MEQ120 (for Glu-120 to Gln mutation), 5`-GAGCATCTGTGCCTTGCT-3`; MEQ168 (for Glu-168 to Gln mutation), 5`-GGATGAACTGTTCCAGGG-3`. Primers T7 and M13 reverse were used as external primers for the polymerase chain reaction. The amplified products were purified by agarose gel electrophoresis and subcloned into pDL1312. The resulting plasmids were sequenced to check the introduction of the mutation. Plasmids encoding mutant neurocalcin with a Glu to Gln change at position 84, 120, or 168 were designated pBNB-MEQ84, pBNB-MEQ120, and pBNB-MEQ168, respectively.

CaBinding Assay

Binding of calcium to neurocalcin or the mutant proteins was determined by ultra filtration. Purified proteins were extensively dialyzed against 25 mM Tris-HCl, pH 8.0, 0.1 M NaCl to remove any traces of EGTA. To remove all the bound calcium, the proteins were then passed through a Ca-chelating column (a kind gift from N. Allbritton) synthesized as described by Meyer et al.(27).

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(2) were placed in the top compartment of Centricon-10 concentrators (molecular mass cut-off of 10 kDa). 20 µl of a 500-µM CaCl(2) solution containing about 10^6 cpm of CaCl(2) 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(2) 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.

Fluorescence Studies

All fluorescence measurements were made on an SLM 8000C spectrofluorimeter (SLM Aminco, Urbana, IL). Tryptophan emission fluorescence spectra of neurocalcins were recorded at 23 °C from 2-ml samples of 3 µM protein in 50 mM MOPS, (^1)pH 7.1, 0.1 M KCl containing either 100 µM CaCl(2) or 2 mM EGTA. The excitation wavelength was set at 290 nm (bandwidth, 4 nm), and the emission wavelength was varied from 300 to 400 nm (bandwidth, 4 nm). Background fluorescence was recorded similarly in absence of added proteins and subtracted from all the spectra. Ca-dependent change of the fluorescence emission of neurocalcins was recorded at 336 nm (bandwidth, 16 nm) in the same conditions as above except that the protein concentration was 1 µM. The free Ca concentration was varied by adding aliquots of EGTA to an initial solution containing 1 mM EGTA, 1.05 mM CaCl(2). The total added volume of EGTA did not exceed 5% of the initial volume. The free Ca concentrations given by these successive additions of EGTA were determined separately by using the fluorescent indicator rhod-2 (Molecular Probes) and a K(d) of 0.57 µM.

Membrane Binding Analysis

Bovine brain membranes were prepared as follows. 5 g of bovine brain tissue were homogenized in 45 ml of 10 mM Hepes, pH 7.5, 0.32 M sucrose, 0.3 mM phenylmethylsulfonyl fluoride, 1 mM EDTA by 15-20 strokes with a Teflon/glass homogenizer. The homogenate was centrifuged at 4 °C for 10 min at 3000 rpm (1,200 times g) in a JA-18 rotor (Beckman). The supernatant was centrifuged at 4 °C for 20 min at 14,000 rpm (29,000 times g) in the same rotor. The pellet was washed by resuspension in the same buffer and recentrifugation. The precipitate from this second centrifugation was suspended in distilled water for 30 min at 4 °C to disrupt synaptosomes. The suspension was centrifuged for 20 min at 8,000 rpm (9,000 times g), and the supernatant was centrifuged again for 20 min at 14,000 rpm (29,000 times g). The pellet (crude synaptic membrane fraction) was resuspended in 10 mM Hepes, pH 7.5, and frozen until used. To trypsin-treat the membranes, 20 µg of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma) were added to 1 ml of brain membranes (100 µg of protein) in 10 mM Hepes, pH 7.5, and incubated for 2 h at 30 °C. 100 µg of soybean trypsin inhibitor (Sigma) were added to stop the reaction. The membranes were pelleted by centrifugation and resuspended in 50 mM MOPS, pH 7.1, 0.1 M KCl.

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(2) (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 times 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 ^3H-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 times 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.

Immunochemistry

Rabbit antisera raised against purified myristoylated neurocalcin were prepared by the Berkeley-Antibody Co. (Berkeley, CA). The antisera reacted primarily with neurocalcin and 10-fold less sensitively with neurocalcin alpha. No cross-reactivity was observed with recombinant myristoylated bovine recoverin (data not shown). 200 µl of crude bovine brain extract (supernatant of 1,200 times g centrifugation) were incubated for 15 min at 22 °C in presence of 1 mM CaCl(2) or 1 mM EGTA and centrifuged for 10 min at 100,000 times g in an airfuge. Supernatant and pellet fractions were electrophoresed on a 15% SDS gel, and proteins were electrotransferred to a nitrocellulose membrane. The membrane was incubated for 30 min at 22 °C in 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5, containing 5% non-fat dried milk and for 2 h with a 1:200 dilution of the anti-neurocalcin antisera in this same buffer. After washing, the bound antibodies were revealed by goat anti-rabbit IgG coupled to alkaline phosphatase (Bio-Rad).


RESULTS

Calcium Binding Properties of Recombinant Neurocalcin

Recombinant myristoylated or unmyristoylated neurocalcin were expressed in E. coli and purified according to a recently described procedure(25) . Fig. 1shows an SDS gel analysis of purified recombinant neurocalcins.


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 (circle) and myristoylated neurocalcin (bullet) in presence of 0.2 mM MgCl(2) and myristoylated neurocalcin in presence of 5 mM MgCl(2) () 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(2), which characterized the binding of the second calcium ion to the protein, is much greater than the constant K(1) 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(2), 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.

Fluorescence of Recombinant Neurocalcin

Neurocalcin has 2 tryptophan residues located at positions 30 and 103. The tryptophan emission spectra of both unmyristoylated and myristoylated recombinant neurocalcin were recorded in the presence and absence of Ca. The binding of Ca induced a 1.5-fold increase in the fluorescence intensity in the case of the unmyristoylated protein and a 2-fold increase for the myristoylated one (Fig. 3A). The emission maximum did not change appreciably. MgCl(2), up to 5 mM, did not induce any change in the fluorescence spectra of these proteins. The dependence of the fluorescence emission intensity at 336 nm on the free Ca concentration is shown in Fig. 3B. The half-maximum change in fluorescence signal of myristoylated neurocalcin was at 1.3-1.7 µM free Ca (five independent determinations) and at 0.25-0.34 µM for the unmyristoylated protein (four independent determinations). A Hill coefficient of 1.8 ± 0.2 for myristoylated neurocalcin indicated that this process was cooperative with respect to Ca. For the unmyristoylated protein, a Hill coefficient of 0.6 ± 0.1 was found. These data show that the two tryptophans of neurocalcin , which are located within the first helix of EF-hands 1 and 3, are sensitive to conformational changes induced by Ca binding and to the presence of a myristic group at the amino terminus of the protein.


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(2) 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 (bullet) or unmyristoylated (circle) 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.



Caand Myristoyl-dependent Binding to Membrane

I examined whether the amino-terminal myristoyl group of recombinant neurocalcins participates in Ca-dependent membrane binding, as was shown for recoverin and hippocalcin. Bovine brain membranes were incubated with either myristoylated or unmyristoylated neurocalcin , in the presence or absence of Ca, and pelleted by high speed centrifugation (100,000 times g). The protein content of both supernatant and pellet were then analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 4). In the presence of Ca, myristoylated neurocalcin was almost completely recovered in the membrane pellet, whereas in the presence of EGTA, it remained in the supernatant. In contrast, the unmyristoylated protein remained in the supernatant fraction whether or not Ca was present. Myristoylated neurocalcin centrifuged at high speed in the presence of Ca but, without membranes, remained fully soluble (data not shown). Hence, the partitioning of myristoylated protein into the membrane in the presence of Ca did not result from a Ca-induced precipitation. Furthermore, when myristoylated neurocalcin was incubated with brain membranes in the presence of 2.5 mM MgCl(2) and 1 mM EGTA (free Ca < 0.05 µM, free Mg > 1.5 mM), no association of the proteins with the membranes could be detected. These data establish that neurocalcin binds to membranes in a Ca- and myristoyl-dependent manner. Furthermore, the nearly complete recovery of myristoylated neurocalcin in the particulate fraction in the presence of Ca shows that the degree of myristoylation of neurocalcin in this preparation was greater than 90%.


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 ^3H-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 ^3H-myristoylated neurocalcin to bovine brain membranes. Membrane binding was determined as described under ``Materials and Methods'' on untreated (bullet) or trypsin-treated (circle) 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).

Calcium Binding Properties of Neurocalcins Harboring Loss-of-function Mutations in Each of the Three Functional Calcium Binding Sites

The relative importance of each of the three functional EF-hands (EF2, EF3, and EF4) in the calcium and membrane binding properties of neurocalcin has been examined using point mutations to inactivate a particular Ca binding site. For this purpose, the conserved glutamate at position 12 of the calcium binding loop, which is critical for binding of calcium as it provides two coordination bonds, was replaced using site-specific mutagenesis by a glutamine (see ``Materials and Methods''). Recombinant myristoylated modified neurocalcins were expressed and purified to homogeneity like the wild-type neurocalcin (Fig. 1). The proteins, disabled in EF2, EF3, or EF4, were designated ME84Q (standing for myristoylated glutamic acid 84 to glutamine-mutated neurocalcin), ME120Q, and ME168Q, respectively.

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: bullet, wild type; , ME84Q (in EF2); box, ME120Q (in EF3); circle, 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.



Fluorescence Properties of Mutant Neurocalcins

The tryptophan emission spectra of the three mutant myristoylated neurocalcins were recorded in the presence and absence of Ca. The three mutant proteins, in the absence of Ca, exhibited essentially the same fluorescence emission spectra as wild-type myristoylated neurocalcin (Fig. 7A). This suggests that the glutamate to glutamine mutation in EF2, EF3, or EF4 did not significantly change the environment of the two tryptophans in the protein. In contrast, the calcium-bound forms of the mutant proteins differed from the calcium-bound wild-type in their fluorescence properties (Fig. 7B). In each case, the binding of calcium changed the quantum yield of tryptophan fluorescence but not the shape of the emission spectra. Mutant ME168Q had the least altered fluorescence properties; the binding of calcium induced a 1.9-fold increase in fluorescence intensity compared to 2.05 for the wild-type protein. Moreover, a plot of the fluorescence change as a function of free calcium (Fig. 8) gave a curve almost identical to that of calcium binding (see Fig. 6). The peak fluorescence intensity of mutant ME120Q increased only 1.3-fold upon binding of calcium. In addition, whereas the two calcium binding sites of ME120Q were nearly saturated at 100 µM free calcium, the change in fluorescence was maximal only above 500 µM. The most pronounced change was observed for mutant ME84Q; the binding of calcium produced a 20% decrease of the maximum fluorescence intensity. This decrease paralleled the binding of calcium (Fig. 6) and reached a plateau at 100 µM free calcium. Interestingly, the fluorescence intensity at (max) of ME84Q increased again with addition of calcium above 500 µM up to 50 mM. This suggests that, at very high concentrations, calcium binds to EF2 of ME84Q despite the Glu to Gln replacement.


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(2) (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.



Membrane Binding Properties of the Mutant Neurocalcins

As shown in Fig. 9, the three myristoylated neurocalcin mutants did bind to membranes in a calcium-dependent manner. For mutants ME120Q and ME168Q, the plot of fraction of protein bound to membranes as a function of free calcium (Fig. 9) was almost identical to the calcium binding curve shown in Fig. 6. This indicates that binding of calcium to EF2 and EF3 (in case of ME168Q) or EF2 and EF4 (in case of ME120Q) was sufficient to promote the specific interaction of the proteins with the membranes. However, this was not the case for mutant ME84Q. As shown in Fig. 9, less than 40% of the protein was associated with the membranes at 200 µM free calcium, although two calcium ions were bound to ME84Q at this concentration (see Fig. 7). Further association of ME84Q with membranes (up to 60% total protein) was observed at higher calcium concentrations. These results indicate that EF2 of neurocalcin plays a critical role in the calcium-induced association of the protein with biological membranes.


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.




DISCUSSION

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, (^2)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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants MH45324 and GM24032 (to L. Stryer). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from CNRS/National Science Foundation. Present address: Département de Biochimie et Génètique Moléculaire, Institut Pasteur, 28, rue du Docteur Roux, F-75 724 Paris-Cedex 15, France. Tel.: 33-1-45-68-83-84; Fax: 33-1-40-61-30-19; Ladant{at}pasteur.fr.

(^1)
The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; ME84Q, ME120Q, and ME168Q, protein designations for myristoylated glutamic acid to glutamine-mutated neurocalcin.

(^2)
D. Ladant, S. Zozulya, and L. Stryer, unpublished observations.


ACKNOWLEDGEMENTS

I am indebted to L. Stryer for constant support in the course of this work and stimulating discussions and critical reading of the manuscript. I thank N. Allbritton, J. Ames, and S. Zozulya for helpful discussions and sharing of materials. I also thank P. Sebo and A. Ullmann for comments on this manuscript.


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