HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 26, 27158-27167, June 25, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/26/27158    most recent M312172200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jeromin, A. Articles by Sharma, Y. Search for Related Content PubMed PubMed Citation Articles by Jeromin, A. Articles by Sharma, Y. Social Bookmarking                      What's this?

N-terminal Myristoylation Regulates Calcium-induced Conformational Changes in Neuronal Calcium Sensor-1^*

Andreas Jeromin, Dasari Muralidhar, Malavika Nair Parameswaran, John Roder¶, Thomas Fairwell||, Suzanne Scarlata**, Louisa Dowal**, Sourajit M. Mustafi, Kandala V. R. Chary, and Yogendra Sharma

From the Neuroscience, Baylor College of Medicine, Houston, Texas 77030, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India, ^¶Mt. Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto, Ontario M5G 1X5, Canada, ^||NHLBI, National Institutes of Health, Bethesda, Maryland 20892, ^**Department of Physiology and Biophysics, Basic Health Sciences Center, State University of New York, Stony Brook, New York 11794-8661, and Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400 005, India

Received for publication, November 6, 2003 , and in revised form, April 5, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Neuronal calcium sensor-1 (NCS-1), a Ca²⁺-binding protein, plays an important role in the modulation of neurotransmitter release and phosphatidylinositol signaling pathway. It is known that the physiological activity of NCS-1 is governed by its myristoylation. Here, we present the role of myristoylation of NSC-1 in governing Ca²⁺ binding and Ca²⁺-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The ⁴⁵Ca binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca²⁺ more strongly (with three Ca²⁺ binding sites) than the non-myristoylated one (with two Ca²⁺ binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca²⁺. Large conformational changes were seen in the near-UV CD with more changes in the case of nonmyristoylated protein than the myristoylated one. Although the changes in the far-UV CD upon Ca²⁺ binding were not seen in E120Q mutant (disabling EF-hand 3), the near-UV CD changes in conformation also were not influenced by this mutation. The difference in the binding affinity of myristoylated and non-myristoylated proteins to Ca²⁺ also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg²⁺-induced changes in near-UV CD are different from Ca²⁺-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca²⁺, which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca²⁺ binding, which might be crucial for its physiological functions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Neuronal Ca²⁺ sensor-1 (NCS-1)¹ belongs to a growing family of EF-hand Ca²⁺-binding proteins, which is conserved from yeast to human (1, 2). The EF-hand is formed by a loop of 12 amino acids flanked by helices E and F (named after helices in parvalbumin), which are positioned like the forefinger and thumb of the right hand (3). This three-dimensional arrangement of the EF-hand motifs in various Ca²⁺-binding proteins is conserved. The proteins of calcium sensor family have in common a fatty acid-modified N terminus, one pseudo or primitive non-canonical EF-hand as the first Ca²⁺ binding site, and three canonical EF-hands.

NCS-1, a 22-kDa protein (theoretical pI 4.7), is a mammalian homologue of frequenin in Drosophila and Xenopus. Frequenin has been shown to enhance synaptic efficacy at the neuromuscular junctions (4, 5). NCS-1 is expressed in neurons and neuromuscular junctions. It also is expressed in other secretory cell types similar to chromaffin cells and in the kidney, suggesting a more generalized role in exocytosis (6–9) that is dependent critically on the spatial and temporal regulation of the intracellular Ca²⁺ concentration (for example see Ref. 10).

The NCS family includes several proteins such as recoverin, neurocalcin, hippocalcin, frequenin, p22, GCAP1, GCAP2, VILIP, and the budding yeast homologue, Frq1. All of them are myristoylated and possess four EF-hand units. The first EF-hand (EF-1) does not bind to Ca²⁺ because of the presence of Gly in place of invariant Asp at the +x position (11). Of all of the aforementioned NCS proteins, recoverin is the most studied protein. It is a Ca²⁺ sensor in retinal rod and cone cells wherein, besides the EF-1, EF-4 is also non-functional and Ca²⁺ binds only to EF-2 and EF-3 sites, leading to the exposure of the buried N-terminal myristoyl group known as Ca²⁺-myristoyl switch (12). Such a movement of the myristoyl group is supposed to expose a hydrophobic pocket that might interact with target proteins and membranes (12, 13). The role of myristoylation is found to be particularly interesting because the molecular mechanisms underlying the physiological role of NCS-1 and its interaction with target proteins such as phosphatidylinositol 4-OH kinase are poorly understood, even though myristoylation is required for the interaction of NCS-1 and phosphatidylinositol 4-OH kinase and up-regulation of its activity (14).

The physiological role of myristoylation varies among the various members of the Ca²⁺ sensor family. For example, the association of p22 with microtubules is dependent both on Ca²⁺ and myristoylation (15), whereas in GCAP1, GCAP2, and VILIP, it is just the Ca²⁺ binding that regulates their function (16, 17). Hippocalcin, neurocalcin, and NCS-1, all of Ca²⁺ sensor family, use their myristoyl group differently. NCS-1 is targeted to membranes via its myristoyl tail (18). In recoverin, the N-terminal myristoylation stabilizes the T conformation (Ca²⁺-free conformation) of the protein, suggesting a role of the myristoyl group as an intrinsic allosteric modulator (19). Based on NMR and fluorescence data, it has been shown that myristoylation does not affect the structure and the Ca²⁺ binding characteristics of yeast Frq1 (20). Further, myristoylated NCS-1 was proposed to be able to bind isolated membranes from chromaffin cells in the absence of Ca²⁺ (6). It plays a more vital role in the regulation of neurotransmitter release and in synaptic plasticity, a highly Ca²⁺-sensitive process (4). NCS-1 not only directly regulates the activity and trafficking of Ca²⁺ channels (21, 22) but also has a direct effect on vesicle availability (23). NCS-1 is implicated in regulating voltage-gated channels (21, 22). In many of these processes, the role of myristoylation and the presence of the Ca²⁺-myristoyl switch in NCS-1 is not defined clearly. Therefore, it is desirable to understand the Ca²⁺ binding properties of NCS-1 and differences between the myristoylated and non-myristoylated forms of the protein. In particular, the data for affinity of mammalianmyristoylated NCS-1 (the biologically active form in a variety of assays) are not available and conflicting results exist for non-myristoylated NCS-1 (24) and yeast Frq1 (20).

In view of these discrepancies, the exact role of myristoylation on the structure and function of NCS-1 is not yet clear. This motivated us to improve an understanding of the structure-function relationships of NCS-1 and, in particular, the role of myristoylation. In this paper, we report the influence of myristoylation on Ca²⁺ binding and Ca²⁺-induced conformational changes in NCS-1 based on various experimental studies. We observe that myristoylation regulates and restricts the Ca²⁺ binding affinity and conformational changes by increasing the cooperativity for ion binding. This is of physiological significance, because Ca²⁺ binding renders these proteins active for their functional roles by introducing conformational changes. Our results suggest the presence of a Ca²⁺-myristoyl switch and that myristoylation influences the Ca²⁺-modulated conformational changes in NCS-1, which might have functional implications. Myristoylated NCS-1 has three Ca²⁺ binding sites with a positive cooperativity among them and a higher apparent affinity compared with non-myristoylated NCS-1, which is found to have just two metal ion binding sites. In addition, the Ca²⁺-myristoyl switch is functional in the E120Q mutant, although other properties such as Ca²⁺-dependent surface hydrophobicity were altered by this mutation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overexpression of Myristoylated and Non-myristoylated NCS-1 and Their Mutants—Overexpression of myristoylated and non-myristoylated NCS-1 was performed following the protocol published previously (25, 26). The rat NCS-1 cDNA was subcloned into pTREC2 (kindly provided by Dr. S. Zozulya, Stanford University) and either transformed alone or co-transformed with pBB131 (kindly provided by Dr. Jeffery Gordon, Washington University, St. Louis, MO) to express N-myristoyltransferase into competent BL21(DE3) cells (Novagen). pTREC2 drives the expression of NCS-1 from the temperature-sensitive phage promoter, whereas the expression of N-myristoyltransferase in pBB131 is dependent on the lacZ promoter. Non-myristoylated NCS-1 was produced by growing cells containing NCS-1 in pTREC2 at 30 °C to an A₆₀₀ of 0.7–1.0 and rapidly shifting the temperature to 42 °C. Myristoylated NCS-1 was produced by growing cells co-transformed with NCS-1 in pTREC2 and pBB131 at 30 °C initially to an A₆₀₀ of 0.2, and 4 mg/liter myristic acid and 12.5 mg/liter sodium myristate were added. N-Myristoyltransferase was expressed by the addition of 1 mM isopropyl--D-thiogalactopyranoside (Roche Applied Science) when A₆₀₀ reached 0.7–1.0. After 30 min, the cells were heat-induced for overexpressing the myristoylated NCS-1 as described above. Cells were harvested by centrifugation and resuspended in 5 ml of buffer (100 mM KCl, 1 mM dithiothreitol (DTT), 1 mM MgCl₂, 50 mM HEPES-K, pH 7.5) per 500 ml of culture medium.

Alternatively, to improve the yield of protein as well as the level of myristoylation, the gene was subcloned in pET21a vector. To produce myristoylated NCS-1, the vector was co-transformed with pBB131 carrying the gene for myristoyltransferase. Protein expression was performed as described above. The data were repeated with proteins purified and prepared in different batches.

E120Q Mutants—The third EF-hand (EF-3) was disabled by creating a site-directed mutation of Glu at position 120 (-z position) by replacing it with Gln (E120Q) using the QuikChange mutagenesis kit (Stratagene). The mutated gene was subcloned in pET21a vector. To produce the myristoylated mutant, the vector was co-transformed with pBB131 to express N-myristoyltransferase. Protein overexpression was performed as described above (26).

Protein Purification by Hydrophobic Interaction Chromatography— Hydrophobic interaction chromatography was performed using prepacked columns and resins obtained from TosoHaas (Montogomeryville, PA) as described previously (26). All of the purifications were done on a Pharmacia fast protein liquid chromatography column with Hewlett-Packard Chemstation software for data acquisition. Large-scale purifications were done on a Toyopearl Phenyl-650C column (0.16 x 20 cm). The sample lysates were loaded in 50 mM HEPES, pH 7.5, containing 100 mM KCl, 1 mM DTT, 1 mM MgCl₂, and 1 mM CaCl₂. A step elution was effected using 50 mM HEPES, pH 7.5, containing 1 mM DTT, 1 mM MgCl₂, and 2 mM EGTA, and a single peak was collected. Alternatively, we have purified proteins on in-house-built columns of phenyl-Sepharose and eluted the protein with 3 mM EGTA after exhaustive washing. The protein purity was checked on SDS-PAGE, and in case of any impurity in the preparation, a final purification was performed on a Superose 12 column attached to a fast protein liquid chromatography.

Biochemical Analysis—The myristoylation of the protein was confirmed by the incorporation of ³H-labeled myristic acid and fluorography (data not shown). Homogeneity of the protein fractions was confirmed by SDS-PAGE, calcium shift assay, and further by Western blotting using mammalian anti-NCS-1 antibodies. Myristoylation was quantitated and analyzed by electrospray ionization mass spectroscopy as described earlier (26). Protein concentration was determined by its absorption at 280 nm using the extinction coefficient of 22,000 M^-1 for NCS-1 (24). Proteins were decalcified immediately before use in the titration. Protein solutions were dialyzed or solvent-exchanged against EDTA followed by calcium-free buffer, which was prepared by passing through a Chelex 100 (Bio-Rad) column and stored in plastic bottles. After complete dialysis, protein solution was passed over a column of Chelex 100 that had been washed exhaustively with MilliQ water followed by the appropriate buffer.

⁴⁵CaCl₂ Binding by Ultrafiltration—Ca²⁺ binding to myristoylated and non-myristoylated NCS-1 was evaluated and compared by the protocol described earlier (27). Protein concentration was 100 µM in both cases. Total calcium concentration was varied from 0 to 500 µM. Samples (500 µl) dissolved in 50 mM Tris buffer, pH 7, containing 100 mM KCl and 1 mM DTT containing ⁴⁵CaCl₂ were loaded on Centricon (Millipore) column and centrifuged for 30 s. Equal aliquots (10 µl) from the retentate and filtrate were used for radioactivity counting for the calculation of free Ca²⁺ concentration in a liquid scintillation counter. Protein and Ca²⁺ concentrations in the column (retentate) were compensated by adding an equal amount of protein. Known concentrations of cold calcium were added in the ultrafiltrate and centrifuged. This was repeated several times, and data were analyzed as shown in Equation 1,

(Eq. 1)

where R_f is the radioactivity in the filtrate, R_p is radioactivity in protein sample, and Ca²⁺(total) and Ca²⁺(free) are the total and free Ca²⁺ concentration, respectively. The experiments were repeated at least three times with the proteins prepared in different batches. Data were analyzed as described by Senin et al. (27) and fitted using the program Origin, version 6.0.

Ca²⁺ Titration by Isothermal Titration Calorimetry (ITC)—ITC measurements with non-myristoylated and myristoylated NCS-1 were performed using a Microcal Omega titration calorimeter. Samples were centrifuged and degassed prior to titration and examined for precipitation, if any, after the titration. In the case of non-myristoylated NCS-1, a typical titration consisted of injecting 1.2-µl aliquots of 10 mM CaCl₂ solution into the protein solution of 0.145 mM (1.43 ml). A total of 47 injections were carried out. In the case of myristoylated NCS-1, the titration was carried out by injecting 2.0-µl aliquots of 10 mM CaCl₂ solution into the protein solution of 0.100 mM (1.43 ml). A total of 40 injections were carried out. In a separate run, the aliquots of the ligand solution were injected into the buffer solution (without the protein) to subtract the heat of dilution. All of the experiments were repeated at least twice. The ITC data were analyzed using Origin (supplied with Omega Micro Calorimeter).

Circular Dichroism Spectroscopy—Circular dichroism spectra were recorded on a Jasco J-715 spectropolarimeter at room temperature. Farand near-UV CD of proteins were performed in 50 mM HEPES, 100 mM KCl, pH 7. To study the effect of cations, proteins were titrated with Ca²⁺ or Mg²⁺ and CD spectra were recorded. Ellipticities were expressed in terms of millidegrees.

Fluorescence Measurements—All of the steady-state fluorescence measurements were done on a Hitachi F-4010 spectrofluorimeter. Emission spectra were recorded in the correct spectrum mode and also corrected for the solvent base line. For quenching experiments, the concentrated stock solutions (5 M) of quencher (potassium iodide and acrylamide) were prepared in water and the protein solution was titrated. The emission spectra were recorded, and the data were analyzed by the Stern-Volmer equation (28) as shown in Equation 2,

(Eq. 2)

or by the modified Stern-Volmer equation as shown in Equation 3,

(Eq. 3)

where F_o and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the molar concentration of quencher (F = F_o - F), K_sv is the Stern-Volmer quenching constant, and f_a gives the fractional accessibility of the NCS-1 fluorophore to quencher.

8-Anilino-1-naphthalene Sulfonic Acid (ANS) Binding—ANS binding experiments were performed by titrating equal amounts of myristoylated and non-myristoylated proteins with ANS (100 µM). Fluorescence spectra were recorded by excitation at 365 nm in the correct spectrum mode. Ca²⁺ or Mg²⁺ was added successively, and the fluorescence was recorded. The spectra were corrected for ANS fluorescence in buffer without protein.

Lipid Vesicle Binding—The binding of myristoylated and non-myristoylated NCS-1 to large unilammellar vesicles of 100 nm in size of palmitoyl oleyl phosphatidylcholine (POPC) and palmitoyl oleyl phosphatidylserine (POPS) in the absence and the presence of Ca²⁺ (100 µM) was performed as described previously by Ehrlich et al. (29) using intrinsic fluorescence or Laurdan-labeled vesicles. The binding was assayed by the change in intrinsic fluorescence upon the binding of the protein to membranes after correction for dilution and the scattering contribution from the lipid. The binding data were normalized using this change.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overexpression, Purification, and Biochemical Characterization of Myristoylated and Non-myristoylated NCS-1 and Its Mutants—Recombinant rat NCS-1 and its myristoylated form were overexpressed in Escherichia coli under the control of a heat-inducible -promoter. Myristoylated NCS-1 was produced by co-expression of N-myristoyltransferase driven by a separate lacZ promoter. The protein was purified at various scales with a single-step purification procedure of hydrophobic interaction chromatography (26). All of the protein preparations were analyzed by SDS-PAGE, Ca²⁺-mobility shift assays, and Western blotting using affinity-purified mammalian anti-NCS-1 antibodies (26). The incorporation of myristoylation was analyzed by electrospray ionization mass spectroscopy. Electrospray ionization mass spectroscopy yielded an average mass of 21,747.79 for the myristoylated NCS-1 and an average mass of 21,533.44 for the non-myristoylated protein, which are in good agreement with their predicted molecular weights of 21,747.47 and 21,537.11, respectively.

To improve the degree of myristoylation of NCS-1, we adopted another approach of overexpression of NCS-1 in the pET-21a vector (Novagen) co-expressed with N-myristoyltransferase. This expression system was more efficient since a higher yield was obtained for both myristoylated and nonmyristoylated NCS-1. With this protocol, the myristoylation level was increased to >90%. Purification was achieved by a single-step chromatography on a phenyl-Sepharose resin. The loss-of-function E120Q mutation was created by site-directed mutagenesis since replacing Glu (at -z position in the EF-3) by Gln, which inactivates this site. The mutant proteins also were purified by the same method with the exception that another step of purification by gel filtration on fast protein liquid chromatography was introduced.

The aim was to identify the role of myristoylation on Ca²⁺ binding properties and protein conformation. For this purpose, we studied Ca²⁺ and Mg²⁺ binding and the Ca²⁺- and Mg²⁺-induced changes in conformation of myristoylated NCS-1 and compared the data with non-myristoylated protein under identical conditions. In addition, the role of N-terminal myristoylation in E120Q mutants (with EF-3 inactive) in influencing the Ca²⁺-myristoyl switch and Ca²⁺-induced conformational changes also was compared.

Myristoylation Increased Cooperativity and Affinity for Ca²⁺, ⁴⁵Ca binding, and ITC Titration—We evaluated the Ca²⁺ binding affinity under identical conditions using the protocol described by Senin et al. (27). All of the parameters such as temperature, protein concentration, volume, and CaCl₂ concentration were controlled carefully by performing the experiments simultaneously. The direct binding assay data showed that the amount of bound Ca²⁺ was greater for myristoylated NCS-1 than for non-myristoylated protein (Fig. 1a). The binding data were evaluated and analyzed as described by Senin et al. (27). The binding sites (n) for myristoylated and non-myristoylated NCS-1 were 3 (n = 2.85 ± 0.15) and 2 (n = 1.8 ± 0.1), respectively (Fig. 1a). Myristoylated NCS-1 showed non-linearity in the Scatchard plot, suggesting a higher degree of cooperativity (Fig. 1b, arrow).

View larger version (23K): [in this window] [in a new window]   FIG. 1. 45Ca binding to non-myristoylated and myristoylated NCS-1. a, non-myristoylated () and myristoylated NCS-1 (•) were incubated with increasing free [Ca2+]. The means ± S.E. are from three independent experiments. b, Scatchard plot for myristoylated protein () and non-myristoylated () NCS-1. The binding experiment was done on the Centricon column using 45CaCl2. Protein concentration used was 100 µM. Arrow at the curvature indicates the high cooperativity.

View larger version (44K): [in this window] [in a new window]   FIG. 2. a, a calorimetric titration of 1.2-µl aliquots of 10 mM CaCl2 solution into 0.145 mM non-myristoylated NCS-1 at 298 K. b, a plot of kcal/mol of heat absorbed/released per injection of CaCl2 as a function of the metal:protein ratio at 298 K and the best least-squares fit of the data to the binding models described. c, a calorimetric titration of 1.2-µl aliquots of 10 mM CaCl2 solution into 0.1 mM myristoylated NCS-1 at 298 K. d, a plot of kcal/mol of heat absorbed/released per injection of CaCl2 as a function of the metal: protein ratio at 298 K and the best leastsquares fit of the data to the binding models described under "Results."

View larger version (28K): [in this window] [in a new window]   FIG. 3. The effect of Ca2+ on far-UV CD of myristoylated protein (a) and non-myristoylated NCS-1 (b) is shown. The additions of calcium were 0, 1, 6, 16, 36, and 66 µM. Protein concentration in each case was 1.33 mg/ml. The effect of Mg2+ on far-UV CD of myristoylated (c) and nonmyristoylated NCS-1 (d) is shown. Mg2+ added were 0, 1, 6, 16, 36, and 66 µM. Protein concentration was 1.33 mg/ml. All of the spectra were recorded in 50 mM Tris buffer, pH 7, containing 100 mM KCl and 1 mM DTT. Path length was 1 cm.

View larger version (24K): [in this window] [in a new window]   FIG. 4. The effect of Ca2+ on near-UV CD of myristoylated protein (a) is shown. Ca2+ added were 0, 2, 7, 12, 22, 32, 52, and 72 µM. b, non-myristoylated NCS-1. Ca2+ added were 0, 2, 12, 22, 32, 42, and 52 µM. Protein concentration in each case was 0.86 mg/ml. The effect of Ca2+ on near-UV CD myristoylated mutant (c) and non-myristoylated (d) mutant is shown. Concentrations of myristoylated and non-myristoylated mutants were 37 and 48 µM, respectively. The effect of Mg2+ on near-UV CD of myristoylated (e) and non-myristoylated (f) NCS-1 is shown. Mg2+ added were 0, 5, 10, 20, and 70 µM. Protein concentration was 1.3 mg/ml. All of the spectra were recorded in 50 mM Tris buffer, pH 7, containing 100 mM KCl and 1 mM DTT in a 1-cm path length cuvette with seven iterations.

Inaccessible and Dynamic Fluorophore in Ca²⁺-bound Myristoylated Protein Monitored by Trp Fluorescence and Potassium Iodide Quenching—Fluorescence spectroscopy was used to ascertain more subtle structural differences between myristoylated and non-myristoylated proteins and the influence of Ca²⁺. There are two Trp residues in the protein, Trp³⁰ in the E helix of EF-1 and Trp¹⁰³ in the E helix of EF-3. Both myristoylated and non-myristoylated NCS-1 showed the emission maximum at 336 nm with 28% less emission intensity for myristoylated protein than for non-myristoylated NCS-1 (Fig. 5a). This is attributed to the restrictions on Trp imposed by the myristoyl group. When myristoylated NCS-1 was titrated with increasing concentrations of Ca²⁺, the fluorescence intensity decreased by 11% (Fig. 5, b and d). The non-myristoylated protein exhibited a similar behavior with the exception that the decrease in emission intensity (8%) (Fig. 5, c and d) was less than that seen in the case of the myristoylated protein (11%). Ca²⁺/protein molar ratios at saturation were 1.23 and 1.07 for non-myristoylated and myristoylated proteins, respectively, indicating a higher affinity for Ca²⁺ for the myristoylated NCS-1. In case of the E120Q mutants, we did not see any change in Trp fluorescence upon Ca²⁺ titration. Mg²⁺ induced only a marginal decrease (2–3%) in myristoylated NCS-1 and an increase (3–4%) in non-myristoylated NCS-1 (data not shown). Mg²⁺ did not induce any changes if added to the Ca²⁺-saturated myristoylated NCS-1. However, Ca²⁺ induced large changes in Mg²⁺-saturated non-myristoylated and myristoylated NCS-1 (data not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 5. Steady-state fluorescence and calcium titration in 50 mM Tris buffer, 100 mM KCl, and 1 mM DTT, pH 7. Protein concentration in both cases was identical (1.4 mg/ml). a, comparison of Trp fluorescence of both proteins under identical conditions. Solid line, myristoylated; dotted line, non-myristoylated protein. b, Ca2+ titration with myristoylated NCS-1. Calcium added were 0, 2, 4, 8, and 14 µM. c, Ca2+ titration with non-myristoylated NCS-1. Ca2+ added were 0, 2, 4, 8, 10, and 16 µM. d, change in the fluorescence emission intensity of non-myristoylated () and myristoylated (•) NCS-1 with Ca2+ titration.

View larger version (17K): [in this window] [in a new window]   FIG. 6. Modified Stern-Volmer plot for the iodide quenching of non-myristoylated and myristoylated NCS-1. a, in the absence of calcium. b, in the presence of 1 mM Ca2+. Protein concentration was 5.5 µM. Experiment was performed in 50 mM Tris buffer, pH 7.5, containing 100 mM KCl and 1 mM DTT.

View larger version (41K): [in this window] [in a new window]   FIG. 7. Change in the fluorescence of ANS and apoprotein complex upon the addition of calcium. a, a comparison of ANS fluorescence of both proteins under identical conditions, a protein concentration of 0.3 mg/ml. Solid line, myristoylated protein; dotted line, non-myristoylated protein. b, Ca2+ titration with non-myristoylated NCS-1. Ca2+ added were 0, 2, 6, 14, and 40 µM. c, Ca2+ titration with E120Q mutant of non-myristoylated protein. Ca2+ added were 2, 4, 14, 34, and 74 µM. d, Ca2+ titration with ANS-myristoylated NCS-1 complex. Ca2+ added were 0, 6, 10, 14, and 20 µM. e, Ca2+ titration with E120Q mutant of myristoylated NCS-1. Ca2+ added were 2, 4, 14, 34, and 74 µM. f, a plot of fluorescence intensity at 476 nm for non-myristoylated protein versus Ca2+ concentration. Change in the actual ANS fluorescence emission upon addition of Ca2+ is shown. Solid line, myristoylated protein; dotted line, non-myristoylated protein.

Effect of Mg²⁺ on ANS Fluorescence—To monitor whether Mg²⁺ can modulate the exposure/solvation of the myristoyl group, the effect of Mg²⁺ on ANS-protein fluorescence was studied. Upon titration with Mg²⁺, the fluorescence intensity decreased concomitantly in myristoylated and non-myristoylated NCS-1 (Fig. 8, a and b) with a greater decrease (41%) in the non-myristoylated protein. In myristoylated NCS-1, the change in ANS fluorescence was comparatively less (7%). Mg²⁺ decreased the ANS fluorescence of E120Q mutants but to a lesser extent than the wild type proteins (Fig. 8, c and d). These results indicate that, although Mg²⁺ was able to induce changes in the protein conformation, it was not able to change the environment of the myristoyl group as Ca²⁺ binding did. Therefore, the solvation or accessibility of myristoyl group is specific to Ca²⁺ and is not Mg²⁺-dependent. In Mg²⁺-saturated ANS-myristoylated protein, the addition of Ca²⁺ increased the ANS fluorescence, confirming the above observations of the solvation of the myristoyl group by Ca²⁺.

View larger version (34K): [in this window] [in a new window]   FIG. 8. Change in the ANS fluorescence upon the addition of Mg2+. a, non-myristoylated NCS-1. b, myristoylated NCS-1. Protein concentration in both cases was 1.15 mg/ml. ANS (100 µM) was added in protein solution in 50 mM Tris buffer, 100 mM KCl, and 1 mM DTT, pH 7, and excited at 285 nm. Mg2+ added were 0, 10, 20, 45, and 110 µM. The effect of Mg2+ on ANS spectra complexed with non-myristoylated (c) and myristoylated (d) mutants is shown. Protein concentration was 6 µM. Ca2+ added were 2, 4, 6, 16, 36 µM.

View larger version (23K): [in this window] [in a new window]   FIG. 9. Normalized change in the fluorescence energy upon POPS and POPC vesicle binding to non-myristoylated NCS-1 (a) and myristoylated NCS-1 (b) in the presence of calcium is shown. The curves of POPC (a) and POPS (b) are the fitted curves.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

⁴⁵Ca binding and ITC experiments with non-myristoylated and myristoylated NCS-1 revealed the number of Ca²⁺ binding sites as two and three, respectively. In the case of myristoylated NCS-1, with three Ca²⁺ binding sites, the binding is highly cooperative. The curvature seen in the Scatchard plot (Fig. 1b) also reflects a greater degree of cooperativity in the case of the myristoylated protein. It is interesting to note that, upon the binding of Ca²⁺ (K₁) to the first site in myristoylated NCS-1, the apparent binding affinity of Ca²⁺ (K₂) to the second site increases almost 1000 times, which is 50 times larger than the apparent binding affinity for the two equal sites in nonmyristoylated NCS-1 (K₂ for myristoylated protein is 1.89 x 10⁷ M^-1, whereas K in the case of the non-myristoylated protein is 5.47 x 10⁵ M^-1). These apparent binding affinities and binding sites measured by ITC were supported by our ⁴⁵Ca binding experiments, also showing the difference in the Ca²⁺ binding affinity of myristoylated and non-myristoylated NCS-1. Our results corroborate the data of Cox et al. (24), which report only two sites but an allosteric effect (1660-fold) for non-myristoylated NCS-1. In comparison, Ames et al. (20) have reported three sites for yeast frg1 under saturation in the absence of Mg²⁺ (K_EF2 = 10 µM; K_EF3/K_EF4 = 0.4 µM). These variations might be the result of the differences in the structure of yeast frq1 and mammalian NCS-1.

The data showing that myristoylation affects Ca²⁺ binding and dynamics were supported by Trp fluorescence and potassium iodide quenching. The two Trp residues of NCS-1 at positions 30 and 103 form part of the E-helices of EF-1 and EF-4. Being in the close vicinity, Trp³⁰ should be relatively more affected by the movement of the myristoyl chain. Quenching of the surface-accessible Trp allows the resolution of the emission spectra of different accessible fluorophores. Potassium iodide quenching shows that, in the presence of Ca²⁺, myristoylation caused inaccessibility of fluorophore. Interestingly, quenching was different when Ca²⁺ was present, indicating a dynamic role played by myristoylation in the presence of Ca²⁺.

We have used ANS fluorescence to study myristoylation-dependent conformational changes in NCS-1. This probe has been used earlier to monitor the presence of the switch by measuring the increase in the accessibility of the Ca²⁺-dependent solvation of recoverin (36). Ca²⁺ binding decreased the ANS fluorescence of non-myristoylated NCS-1 and increased the fluorescence of the myristoylated protein. This increased ANS fluorescence upon binding Ca²⁺ seen in the case of myristoylated NCS-1 reflects the possible exposure of the myristoyl group, which becomes accessible to ANS binding in the presence of Ca²⁺. Mg²⁺ was not able to increase the ANS fluorescence of myristoylated NCS-1 as Ca²⁺ did, indicating the myristoyl-sensitive conformational changes by Ca²⁺ and not by Mg²⁺. In both non-myristoylated and myristoylated E120Q mutants, the ANS fluorescence was increased in the presence of Ca²⁺ with a greater increase in the myristoylated mutant. This indicates that myristoylation affects the packing and hydrophobic properties in relation to Ca²⁺ binding. The effect of Mg²⁺ on both mutants has not been very different from the non-myristoylated NCS-1 mutant, indicating that Mg²⁺ binding properties were not altered by the E120Q mutation.

Ca²⁺-dependent binding of myristoylated NCS-1 to POPC also indicates the possible extrusion of myristoyl chain from the protein core, although the binding to other lipids was not enhanced significantly in the presence of Ca²⁺. Because of the complex nature of the interaction of NCS-1 with membranes, this does not allow us to explain the preference for particular lipid vesicles. The membrane interaction of NCS-1 is not likely to be mediated by electrostatic interactions, because there is no net charge on the N terminus. However, we can suggest that myristoylation facilitates the binding of NCS-1 to specific membranes and thus allows interactions with target proteins. The interaction of NCS-1 with phosphatidylinositol 4-OH kinase and its modulation is dependent on myristoylation (37). Myristoylation of NCS-1 is important for the direct interaction with phosphatidylinositol 4-OH kinase and up-regulation of its activity, corroborating the importance of myristoylation for regulating the physiological activity of NCS-1. Interestingly, the myristoylation mutant of yeast Frq1 is also less efficient in rescuing the phenotype of the null mutant, suggesting that myristoylation is important for the biological activity of yeast Frq1 as well (10).

In conclusion, our data show that the myristoylation influences the Ca²⁺ binding. We believe that related Ca²⁺-binding proteins would show a similar pattern in their myristoylated form with respect to Ca²⁺ binding. In fact, Ladant (38) has made similar observations of the restricted conformational changes in myristoylated neurocalcin whose crystal structure is similar to the human non-myristoylated NCS-1 structure (39). It will be important to determine how the myristoylation-restricted calcium-induced changes in conformation and affinity relate to the various physiological roles of these calcium sensors/calcium-binding proteins. Determining the solution structure of myristoylated NCS-1 will help dissect out common principles that govern the structure-function relationships of these proteins. This is of utmost importance, because the release of the neurotransmitter and exocytosis in general is regulated strongly by the intracellular calcium concentration.

	FOOTNOTES

 
^* This work was supported by the Medical Research Council of Canada (to A. J. and J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Fax: 91-40-271-60-591; E-mail: yogendra{at}ccmb.res.in.

¹ The abbreviations used are: NCS-1, neuronal calcium sensor-1; CD, circular dichroism; ANS, 8-anilino-1-naphthalene sulfonic acid; DTT, dithiothreitol; ITC, isothermal titration calorimetry; POPC, palmitoyl oleyl phosphatidylcholine; POPS, palmitoyl oleyl phosphatidylserine.

	ACKNOWLEDGMENTS

 
We thank A. Amrutha for help in some of the experiments and Dr. T. Ramakrishna for helpful discussion and Sayeed Syed Abdul for technical assistance. We thank Dr. Karl-W. Koch (Institut für Biologische Informationsverarbeitung) for providing the protocol for calcium binding experiments.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Braunewell, K. H., and Gundelfinger, E. D. (1999) Cell Tissue Res. 295, 1-12[CrossRef][Medline] [Order article via Infotrieve]
2. Burgoyne, R. D., and Weiss, J. L. (2001) Biochem. J. 353, 1-12[CrossRef][Medline] [Order article via Infotrieve]
3. Kretsinger, R. H. (1975) in Calcium Transport in Contraction and Secretion (Carafoli, E., Clementi, F., Drabikowski, W., and Margeth, A., eds) pp. 469-478, North Holland Publishing Co., Amsterdam
4. Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J., Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron 11, 15-28[CrossRef][Medline] [Order article via Infotrieve]
5. Olafsson, P., Wang, T., and Lu, B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8001-8005[Abstract/Free Full Text]
6. McFerran, B. W., Graham, M. E., and Burgoyne, R. D. (1998) J. Biol. Chem. 273, 22768-22772[Abstract/Free Full Text]
7. McFerran, B. W., Weiss, J. L., and Burgoyne, R. D. (1999) J. Biol. Chem. 274, 30258-30265[Abstract/Free Full Text]
8. Weisz, O. A., Gibson, G. A., Leung, S. M., Roders, J., and Jeromin, A. (2000) J. Biol. Chem. 275, 24341-24347[Abstract/Free Full Text]
9. Werle, M., Roder, J., and Jeromin, A. (2000) Neurosci. Lett. 284, 33-36[CrossRef][Medline] [Order article via Infotrieve]
10. Rettig, J., and Neher, E. (2002) Science 298, 781-785[Abstract/Free Full Text]
11. Tanaka, T., Ames, J. B., Harvey, T. S., Stryer, S., and Ikura, M. (1995) Nature 376, 444-447[CrossRef][Medline] [Order article via Infotrieve]
12. Ames, J. B., Ishima, R., Tanaka, T., Gordon, J. I., Stryer, L., and Ikura, M. (1997) Nature 389, 198-202[CrossRef][Medline] [Order article via Infotrieve]
13. Zozulya, S., and Streyer, L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11569-11573[Abstract/Free Full Text]
14. Zhao, X., Varnai, P., Tuymetova, G., Balla, A., Toth, Z. E., Oker-Blom, C., Roder, J., Jeromin, A., and Balla, T. (2001) J. Biol. Chem. 276, 40183-40189[Abstract/Free Full Text]
15. Timm, S., Titus, B., Bernd, K., and Barroso, M. (1999) Mol. Biol. Cell. 10, 3473-3488[Abstract/Free Full Text]
16. Braunewell, K. H., Spilker, C., Behnisch, T., and Gundelfinger, E. D. (1997) J. Neurochem. 68, 2129-2139[Medline] [Order article via Infotrieve]
17. Olshevskaya, E. V., Hughes, R. E., Hurley, J. B., and Dizhoor, A. M. (1997) J. Biol. Chem. 272, 14327-14333[Abstract/Free Full Text]
18. O'Callaghan, D. W., Ivings, L., Weiss, J. L., Ashby, M. C., Tepikin, A. V., and Burgoyne, R. D. (2002) J. Biol. Chem. 277, 14227-14237[Abstract/Free Full Text]
19. Weiergraber, O. H., Senin, I. I., Philippov, P. P., Granzin, J., and Koch, K. W. (2003) J. Biol. Chem. 278, 22972-22979[Abstract/Free Full Text]
20. Ames, J. B., Hendricks, K. B., Strahl, T., Huttner, I. G., Hamasaki, N., and Thorner, J. (2000) Biochemistry 39, 12149-12161[CrossRef][Medline] [Order article via Infotrieve]
21. Tsujimoto, T., Jeromin, A., Saitoh, N., Roder, J. C., and Takahashi, T. (2002) Science 295, 2276-2279[Abstract/Free Full Text]
22. Rousset, M., Cens, T., Gavarini, S., Jeromin, A., and Charnet, P. (2003) J. Biol. Chem. 278, 7019-7026[Abstract/Free Full Text]
23. Sippy, T., Cruz-Martin, A., Jeromin, A., and Schweizer, F. E. (2003) Nat. Neurosci. 6, 1031-1038[CrossRef][Medline] [Order article via Infotrieve]
24. Cox, J. A., Durussel, I., Comte, M., Nef, S., Nef, P., Lenz, S. E., and Gundelfinger, E. D. (1994) J. Biol. Chem. 269, 32807-32813[Abstract/Free Full Text]
25. Zozulya, S., Ladant, D., and Streyer, L. (1995) Methods Enzymol. 250, 383-393[Medline] [Order article via Infotrieve]
26. Fisher, J. R., Sharma, Y., Iuliano, S., Piccioti, R. A., Krylov, D., Hurley, J., Roder, J., and Jeromin, A. (2000) Protein Expression Purif. 20, 66-72[CrossRef][Medline] [Order article via Infotrieve]
27. Senin, I. I., Fischer, T., Komolov, K. E., Zinchenko, D. V., Philippov, P. P., and Koch, K. W. (2002) J. Biol. Chem. 277, 50365-50372[Abstract/Free Full Text]
28. Lakowicz (1983) Principles of Fluorescence Spectroscopy, Plenum Press, New York
29. Ehrlich, L. S., Fong, S., Scarlata, S., Zybarth, G., and Carter, C. (1996) Biochemistry 35, 3933-3943[CrossRef][Medline] [Order article via Infotrieve]
30. Wiseman, T., Williston, S., Brandts, J. F., and Lin, L. N. (1989) Anal. Biochem. 79, 131-137
31. Schellman, J. A., and Oriel, P. (1962) J. Chem. Phys. 37, 2114-2124[CrossRef]
32. Woody, R. W., and Tinoco, I. (1967) J. Chem. Phys. 46, 4927-4945[CrossRef]
33. Fasman, G. D. (1996) Circular Dichroism and Conformational Analysis of Biomolecules, Plenum Press, New York
34. Bhatnagar, R. S., Futterer, K., Waksman, G., and Gordon, J. I. (1999) Biochim. Biophys. Acta 1441, 162-172[Medline] [Order article via Infotrieve]
35. Kawamura, S., Hisatomi, O., Kayada, S., Tokunaga, F., and Kuo, C. H. (1993) J. Biol. Chem. 268, 14579-14582[Abstract/Free Full Text]
36. Ames, J. B., Porumb, T., Tanaka, T., Ikura, M., and Stryer, L. (1995) J. Biol. Chem. 270, 4526-4533[Abstract/Free Full Text]
37. Hughes, R. E., Brozovic, P. S., Klevit, R. E., and Hurley, J. B. (1995) Biochemistry 34, 11410-11416[CrossRef][Medline] [Order article via Infotrieve]
38. Ladant, D. (1995) J. Biol. Chem. 270, 3179-3185[Abstract/Free Full Text]
39. Bourne, Y., Dannenberg, J., Pollmann, V., Marchot, P., and Pongs, O. (2001) J. Biol. Chem. 276, 11949-11955[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg