©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amino-terminal Myristoylation Induces Cooperative Calcium Binding to Recoverin (*)

(Received for publication, October 21, 1994; and in revised form, December 12, 1994)

James B. Ames (1)(§) Tudor Porumb (2) Toshiyuki Tanaka (2)(¶) Mitsuhiko Ikura (2)(**)(§§) Lubert Stryer (1)(§§)

From the  (1)Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305 and the (2)Division of Molecular and Structural Biology, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Recoverin, a new member of the EF-hand protein superfamily, serves as a Ca sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to bind to disc membranes when the Ca level is elevated. Ca-bound recoverin prolongs the lifetime of photoexcited rhodopsin, most likely by blocking its phosphorylation. We report here Ca binding studies of myristoylated and unmyristoylated recombinant recoverin using flow dialysis, fluorescence, and NMR spectroscopy. Unmyristoylated recoverin exhibits heterogeneous and uncooperative binding of two Ca with dissociation constants of 0.11 and 6.9 µM. In contrast, two Ca bind cooperatively to myristoylated recoverin with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 µM. Thus, the attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity in Ca binding. One-dimensional ^1H and two-dimensional N-^1H shift correlation NMR spectra of myristoylated recoverin measured as a function of Ca concentration show that a concerted conformational change occurs when two Ca are bound. The Ca binding and NMR data can be fit to a concerted allosteric model in which the two Ca binding sites have different affinities in both the T and R states. The T and R conformational states are defined in terms of the Ca-myristoyl switch; in the T state, the myristoyl group is sequestered inside the protein, whereas in the R state, the myristoyl group is extruded. Ca binds to the R state at least 10,000-fold more tightly than to T. In this model, the dissociation constants of the two sites in the R state of the myristoylated protein are 0.11 and 6.9 µM, as in unmyristoylated recoverin. The ratio of the unliganded form of T to that of R is estimated to be 400 for myristoylated and <0.05 for unmyristoylated recoverin. Thus, the attached myristoyl group has two related roles: it shifts the T/R ratio of the unliganded protein more than 8000-fold, and serves as a membrane anchor for the fully liganded protein.


INTRODUCTION

Recoverin is a 23-kDa calcium-binding protein in retinal rod and cone cells. The Ca-bound form of recoverin prolongs the photoresponse (Gray-Keller et al., 1993), most likely by blocking the phosphorylation of photoexcited rhodopsin (Kawamura, 1993). The light-induced lowering of the Ca level in rod cells is thought to reverse this inhibition, thereby promoting recovery of the dark state. The shortening of the lifetime of photoexcited rhodopsin at low Ca concentration may also contribute to adaptation to background light. Calcium ion is known to play a critical role in adaptation (Koch, 1994; Koch and Stryer, 1988; Matthews et al., 1988).

Retinal recoverin contains a covalently attached N-terminal myristoyl or related fatty acyl group (Dizhoor et al., 1992). Ca binding to recoverin is thought to induce the extrusion of the myristoyl group of the protein, enabling it to interact with the disc membranes (Dizhoor et al., 1993; Zozulya and Stryer, 1992). This Ca-induced membrane interaction, referred to as a ``Ca-myristoyl switch,'' may be important in regulating the phosphorylation of photoexcited rhodopsin.

Sequence comparison of recoverin with other members of the EF-hand protein superfamily has revealed the existence of four EF-hand Ca binding motifs (Ray et al., 1992). Fig. 1shows the amino acid sequence of the four EF-hands of recoverin. The Ca-coordinating residues at positions 5, 9, and 12 of the 12-residue loop are well preserved for all of the four motifs. However, EF-1 and EF-4 lack essential acidic residues at positions 1 and 3. Thus, EF-2 and EF-3, but not EF-1 and EF-4, are predicted to have high affinity for Ca.


Figure 1: Primary sequence of the four EF-hand motifs found in recoverin. The EF-hand consensus sequence is shown at the top with Ca ligating residues denoted as X, Y, and Z; n is any nonpolar residue; and a dash is any residue. Conserved amino acid residues in the Ca binding loop and three tryptophans are in boldface.



The crystal structure of recoverin shows that it in fact contains four EF-hands (Flaherty et al., 1993). However, the structure of recoverin is significantly different from that of calmodulin (Babu et al., 1988) and troponin C (Herzberg and James, 1988). In calmodulin and troponin C, two EF-hand pairs are separated by a long central helix, forming a dumbbell-shaped structure in the crystal. Recoverin adopts a compact globular shape that can be divided into amino- and carboxy-terminal domains. The amino-terminal domain contains EF-1 and EF-2, and the carboxyl-terminal domain contains EF-3 and EF-4. Ca was found to bind to EF-3 in this crystal form, and Sm, used as an isomorphous heavy atom derivative, was found to bind to both EF-2 and EF-3. EF-1 is distorted from a favorable Ca-binding geometry by Cys-39 and Pro-40 at positions 3 and 4 of the 12-residue loop. EF-4 contains a salt bridge between Lys-161 and Glu-171 at positions 2 and 12. The crystal contained no metal ion in EF-1 and EF-4.

Extensive Ca binding studies have been carried out on the EF-hand proteins calmodulin (Haiech et al., 1981; Minowa and Yagi, 1984; Klee, 1988; Linse et al., 1991; Porumb, 1994), troponin C (Iida, 1988), parvalbumin (Ogawa and Tanokura, 1986), and calbindin-D (Akke et al., 1991). In calmodulin, two high affinity Ca binding sites are present in the carboxyl- terminal domain, and two low affinity sites are present in the amino-terminal domain. Cooperativity was observed for the pair of sites in each domain, but not between the amino- and carboxyl-terminal domains (Minowa and Yagi, 1984). However, little is known about calcium binding to recoverin. To understand the molecular mechanism of the Ca-myristoyl switch, a thorough knowledge of the calcium binding properties of recoverin is necessary. We report here flow dialysis, fluorescence, and NMR studies of myristoylated and unmyristoylated recoverin that were carried out to delineate the mechanism of Ca binding and gain insight into the mechanism of the calcium-myristoyl switch.


MATERIALS AND METHODS

Sample Preparation

Recombinant myristoylated and unmyristoylated recoverins were expressed in the overproducing Escherichia coli strains pTrec2/pBB131/DH5alphaF and pTrec2/DH5alpha, respectively, and were purified as described previously (Zozulya and Stryer, 1992). Myristic acid (5 mg/ml in ethanol) was added to the medium 1 h before induction to give a final concentration of 5 mg/liter. Uniformly N-labeled protein with an unlabeled myristoyl group covalently attached to the amino terminus was obtained by growing E. coli cells carrying pTrec2 and pBB131 plasmids in M9 minimal medium as described previously (Ames et al., 1994).

Tryptophan fluorescence experiments were performed with 1-2 µM myristoylated or unmyristoylated recoverin in 2 ml of 0.1 M KCl, 1 mM dithiothreitol, 50 mM HEPES (pH 7.4) at 25 °C. The free calcium concentration (50 nM to 2 µM) was set using an EGTA buffer system. The protein samples initially contained an equal amount of total Ca and EGTA (2 mM); the free Ca concentration was adjusted by adding aliquots of 0.1 M EGTA. The free Ca concentration was calculated based on the total amount of Ca and EGTA present using the program Bound-and-Determined (Brooks and Storey, 1992). The calculated free calcium concentrations agreed closely with measured Ca concentrations using fluorescent indicator dyes fluo-3 and rhod-2 (Molecular Probes, Eugene, OR) with K(d) of 0.4 and 1.0 µM, respectively (Tsien and Pozzan, 1989). Calcium concentrations above 1 µM were measured using a Ca-selective electrode (Orion, Boston, MA).

Samples for NMR experiments were prepared by dissolving lyophilized protein (1 mM) in 95% H(2)O, 5% ^2H(2)O or 99.9% ^2H(2)O (total volume, 500 µl each) containing 0.1 M KCl and 10 mM [^2H]dithiothreitol. The pH was adjusted to 6.8-7.0 using 4% NaO^2H or 4% ^2HCl without correction for isotope effects. The total calcium concentration of the NMR samples was adjusted by successively adding 1.0-1.5-µl aliquots of 0.1 M CaCl(2).

Flow Dialysis

Ca-binding curves were obtained by flow dialysis using a home-built apparatus as described previously (Porumb, 1994). The dialysis medium contained 0.2 mM myristoylated or unmyristoylated recoverin in 0.1 M KCl, 50 mM HEPES-KOH (pH 7.5) at 25 °C. The Ca-binding data were quantitatively analyzed using the classical Hill, Scatchard, and multiple binding site equations (Dahlquist, 1979). Nonlinear regression was performed using the computer program Origin (MicroCal Software, Inc., Northampton, MA) and extensions of the Enzfitter computer program (Leatherbarrow, 1987).

Fluorescence Spectroscopy

Steady state fluorescence and emission spectra were measured on an SLM 8000C spectrofluorimeter (SLM Aminco, Urbana, IL). The excitation wavelength was 290 nm for recoverin. Emission spectra of recoverin as a function of Ca were recorded from 300 to 410 nm, and a buffer blank was subtracted. The relative sensitivity of the detection system was 1.00, 0.98, 1.09, 1.13, and 1.46 at 300, 325, 350, 375, and 400 nm, respectively. For quantitative Ca titrations, undispersed fluorescence was measured through long pass filters (Schott WG-345).

NMR Spectroscopy

One-dimensional (1D) (^1)^1H NMR spectra of myristoylated and unmyristoylated recoverin as a function of Ca were recorded on a GENMR-500 spectrometer. The spectral width was 6000 Hz with digital resolution of 0.75 Hz. All spectra were recorded with 200 transients.

Two-dimensional NMR experiments were performed on a UNITY-plus 500 spectrometer equipped with a triple resonance probe with an actively shielded z gradient together with a pulse field gradient accessory. N-^1H HSQC (Bodenhausen and Ruben, 1980; Bax et al., 1990) spectra as a function of Ca were recorded using the enhanced sensitivity method of Kay et al.(1992). The spectra were recorded with the following numbers of complex points and acquisition times: N (F(1)) 128, 64 ms, ^1H (F(2)) 512, 77 ms. The HSQC spectrum of the Ca-free state was recorded with 64 transients. Spectra with 0.75, 1.5, 2.25, 3.0, 4.0, and 5.0 equivalents of Ca were recorded with eight transients.

All data sets were processed on a Sun Sparc10 workstation using commercial software (NMRZ, New Methods Research Inc., Syracuse, NY; and FELIX, Biosym, San Diego, CA). The programs Capp and Pipp (Garrett et al., 1991) were used for peak selection and spectral analysis. Residual water signal in N-^1H HSQC spectra was removed using a time-domain deconvolution approach reported by Marion et al.(1989).


RESULTS

Measurements of Ca Binding

Ca binding curves of myristoylated and unmyristoylated recoverin (Fig. 2A) were measured by the flow dialysis method. About two Ca are bound at saturation. Unmyristoylated recoverin exhibits markedly biphasic Ca binding; the Hill coefficient at half-saturation is 0.4. It is evident that unmyristoylated recoverin contains two Ca binding sites that have markedly different affinities. The heterogeneity and lack of cooperativity in Ca-binding is also seen in the biphasic Scatchard plot (Fig. 2B).


Figure 2: A, Ca binding curves of myristoylated (filledsquares) and unmyristoylated (opencircles) recoverin obtained by flow dialysis. The solidlines represent the best fit to the allosteric model presented in Fig. 4(see Table 1for refined parameters). The crossedpoints show the relative change in fluorescence intensity of myristoylated recoverin as a function of Ca concentration. B, Scatchard plots of Ca binding data of myristoylated (lefty axis) and unmyristoylated (righty axis) recoverin. The calcium binding curves measured by flow dialysis appear to saturate at about two Ca bound per protein (2.3 for myristoylated and 1.6 for unmyristoylated). The NMR data presented below clearly demonstrate that in fact 2 ± 0.15 Ca bind to recoverin. Thus, a Ca stoichiometry of two was used to normalize the Ca binding curves in Fig. 2A.




Figure 4: Allosteric model for the binding of Ca to recoverin. The equilibrium expressions and parameters are defined in the text.





The binding equilibria for two different Ca-binding sites are

where [Ca](f) is the free calcium concentration, P denotes the occupancy of sites i and j, and the K(D) values are the dissociation constants. Both sites are empty in P, one is filled in P and P, and both are filled in P. The fractional occupancy Y of the calcium binding sites is given by

If the sites are independent (K(D)(1) = K(D)(4) and K(D)(2) = K(D)(3)), the binding expression simplifies to

The binding data for unmyristoylated recoverin are well fit by this equation with K(D)(1) = 0.11 µM and K(D)(2) = 6.9 µM. As discussed below, it seems likely that EF-3 is the higher affinity site and EF-2 is the lower affinity site (Flaherty et al., 1993). P, then, denotes occupancy of EF-3, and P denotes occupancy of EF-2.

The binding curve for myristoylated recoverin, in contrast with that of the unmyristoylated protein, exhibits a steep Ca dependence. The data for the myristoylated protein can be fit to the Hill equation

with a Hill coefficient (alpha) of 1.75 and an apparent dissociation constant (K(d)) of 17.4 µM (Table 1). The observed Hill coefficient of 1.75 requires that two or more Ca bind cooperatively. Cooperativity is also demonstrated by the downward curvature seen in the Scatchard plot (Fig. 2B). The binding curve of myristoylated recoverin was also fit by the two-site model described above, yielding K(D)(1) = 100 µM, K(D)(2) = 2.4 µM, K(D)(3) = 2.4 mM, and K(D)(4) = 0.1 µM.

The dependence of the fraction f of P, P, P, and P as a function of [Ca](f) was calculated using the best-fit parameters of the two-site model and plotted in Fig. 3. For unmyristoylated recoverin (Fig. 3A), f builds up to a maximum of about 0.79 as [Ca](f) is raised to 0.85 µM and then falls as the Ca concentration is raised. The fraction of the other one-calcium species, f, is always less than 0.014. Thus, Ca binds almost exclusively to the first site of the unmyristoylated protein before binding to the second. By contrast, for myristoylated recoverin (Fig. 3B), the maximum value of f is less than 0.08. In fact, the fraction of molecules in the P state closely parallels Y, showing that the binding of Ca to myristoylated recoverin is highly cooperative.


Figure 3: Concentration profiles of protein states of (A) unmyristoylated and (B) myristoylated recoverin with zero, one, and two Ca bound for a two-site model. The fractional concentrations of the protein states are defined as f, where the subscripts i and j denote whether or not Ca is bound to the low affinity (EF-2) and high affinity (EF-3) sites, respectively. The data points represent the fractional saturation of myristoylated recoverin measured by flow dialysis. The solidlines represent concentration profiles calculated for a two-site model (see text).



The concerted allosteric model (Monod-Wyman-Changeux model by Monod et al.(1965)) can be adapted to quantitatively account for both the uncooperative binding of Ca by unmyristoylated recoverin and the cooperative binding by myristoylated recoverin. In the model depicted in Fig. 4, recoverin has two intrinsically different binding sites for calcium and two global protein conformational states, T and R. Let K(1) and K(2) denote the dissociation constants of the two sites (most likely EF-3 and EF-2, respectively) in the R state. We assume that the global conformational transition from R to T changes the binding constant of both sites by the same factor c. L is the T/R ratio in the absence of Ca. The equilibria for this model are given by

where R and T represent recoverin in the R and T states, and the subscripts i and j denote whether or not Ca is bound to the low affinity (EF-2) and high affinity (EF-3) sites, respectively. The fractional saturation is then given by

where beta = c + L and = c^2 + L. The dissociation constants of the R state (K(1) and K(2)) are taken to be equal to K(D)(1) and K(D)(2) obtained by the independent sites model for unmyristoylated recoverin. Using K(1) = 0.11 µM and K(2) = 6.9 µM, L is calculated to be 400 for myristoylated and leq0.05 for unmyristoylated recoverin. The parameter c is estimated from the data for the myristoylated form to be less than 10, indicating that Ca binds almost exclusively to the R state.

Both cooperative and uncooperative Ca binding can be modeled by varying L while keeping K(1), K(2), and c fixed (Fig. 2A). Positive cooperativity is produced when T is thermodynamically favored over R (L > 1) and Ca binds more tightly to R than to T (c = 10). In the absence of the myristoyl group, the R state is much more stable. The myristoyl group induces cooperative Ca binding by markedly favoring T. The myristoyl group changes the T/R equilibrium by a factor of at least 8000 (400/0.05). Thus, the myristoyl group serves as a covalently attached allosteric effector.

Ca-induced Conformational Changes

NMR Spectroscopy

Protein conformational changes resulting from Ca binding were monitored by NMR spectroscopy. Because the concentration of recoverin (1 mM) was much higher than the dissociation constants of the calcium binding sites, nearly all of the added Ca was bound, up to two Ca/recoverin. The dependence of the 1D ^1H NMR spectra of myristoylated and unmyristoylated recoverin on the number of bound Ca is shown in Fig. 5. Peaks in the aliphatic (-1-2 ppm) and aromatic (6-8 ppm) regions of the spectrum are the most revealing indices of Ca-induced structural changes. Fig. 6A shows intensities of selected peaks from 1D H NMR spectra of unmyristoylated recoverin as a function of added Ca. The intensity of the -0.47 ppm line saturates at nearly 1 equivalent of bound Ca. This line monitors Ca binding to the first site and is unaffected by Ca binding to the second site. In other words, the intensity of the -0.47 ppm line increases in forming P and stays the same in forming P. A different Ca dependence is seen for the 5.61-ppm line, which increases to a maximum as the number of bound Ca increases from 0 to 1 (in going from P to P), and then falls to the base line as the number of bound Ca increases from 1 to 2 (in going from P to P). Hence, the 5.61-ppm line is responsive to Ca binding to both sites. The 5.61-ppm line profile indicates that two Ca bind to unmyristoylated recoverin in a sequential rather than concerted manner, consistent with the flow dialysis data on unmyristoylated recoverin (Fig. 2) and the calculated proportion of the P species (Fig. 3A).


Figure 5: 1D ^1H NMR spectra of (A) unmyristoylated and (B) myristoylated recoverin at various equivalents of added Ca. Intensities of highlighted peaks are plotted in Fig. 6.




Figure 6: Intensities of 1D ^1H NMR peaks of (A) unmyristoylated recoverin and (B) myristoylated recoverin as a function of Ca. It should be noted that the x axis represents Ca added/recoverin and not free calcium concentration as in Fig. 2A. Since the concentration of recoverin (1 mM) used in the NMR experiments is 100-fold greater than the Ca dissociation constant (K = 17 µM), all Ca added, up to two equivalents, was bound to the protein.



Fig. 6B shows selected Ca-dependent peak intensities from 1D ^1H NMR spectra of myristoylated recoverin. The peak intensities changed monotonically on the addition of up to two equivalents of Ca (in going from P to P). No further change was detected beyond 2 bound Ca/recoverin. This dependence shows that 2 ± 0.15 calcium ions bind per protein, in agreement with the flow dialysis measurements. None of the resolved peak intensities saturated at 1 equivalent of Ca. The absence of detectable intermediate Ca binding states supports a highly cooperative Ca binding mechanism for myristoylated recoverin ( Fig. 2and Fig. 3B). A similar phenomenon has been observed for the high affinity sites of calmodulin (Ikura et al., 1983; Klevit et al., 1984; Thulin et al., 1984) and troponin C (Tsuda et al., 1988).

Two-dimensional N-^1H HSQC spectra of uniformly N-labeled myristoylated recoverin were obtained as a function of Ca. Complete sequence-specific backbone assignments have been obtained for myristoylated recoverin in the Ca-free state (Ames et al., 1994). This enables one to monitor specific residues that undergo structural changes upon Ca binding. The N-^1H HSQC spectral changes are so substantial upon the addition of Ca that very few peaks coincide in the spectra of the Ca-free and Ca-bound states (Fig. 7). N-^1H cross-peak intensities of backbone amide and tryptophan imide protons of specific residues in the Ca-free state are plotted as a function of bound Ca (Fig. 8). Cross-peaks from residues in the amino-terminal domain and also in the carboxyl-terminal domain decrease monotonically to zero when the number of bound Ca increases from 0 to 2. These data indicate that Ca-induced structural changes occur throughout the protein in a concerted fashion.


Figure 7: Two-dimensional N-^1H HSQC NMR spectra of (A) Ca-free and (B) Ca-bound myristoylated recoverin uniformly labeled with N (>95%). CHAPS detergent (20 mM) was added to prevent aggregation of the Ca-bound state (Kataoka et al., 1993). The addition of CHAPS decreased the linewidths (Anglister et al., 1993) but did not change the peak positions of the cross-peaks in ^1H-N HSQC spectrum. Therefore, the addition of CHAPS most likely has little effect on the structure of recoverin.




Figure 8: Two-dimensional N-^1H HSQC cross-peak intensities of selected residues in the amino- and carboxyl-terminal domains of myristoylated recoverin at various equivalents of added Ca.



Fluorescence Spectroscopy

Recoverin contains three tryptophan residues (Trp-31 in EF-1, Trp-104 in EF-3, and Trp-156 in EF-4) whose fluorescence emission is responsive to Ca binding (Dizhoor et al., 1991). The fluorescence emission spectrum of myristoylated recoverin shifts to the red by 7 nm when Ca binds to recoverin (Ray et al., 1992). The integrated emission intensity at wavelengths longer than 350 nm increases 2-fold when Ca binds to myristoylated recoverin. This change was used to monitor Ca-induced protein structural changes. The crossed points in Fig. 2A show the dependence of the integrated fluorescence intensity on the free Ca concentration. The normalized fluorescence intensity profile nearly coincides with the Ca binding curve determined by flow dialysis. Analysis of the fluorescence profile using the Hill equation yields a Hill coefficient of 1.80 and K(d) of 15 µM, in good agreement with the values obtained by flow dialysis. These fluorescence results provide further evidence that a cooperative protein structural change occurs when two Ca bind to myristoylated recoverin.


DISCUSSION

The calcium binding properties of recoverin were determined to help understand how the Ca-myristoyl switch operates. Many differences between the Ca binding properties of the myristoylated and unmyristoylated protein are evident. Myristoylated recoverin cooperatively binds two Ca with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 µM. Unmyristoylated recoverin, by contrast, binds two Ca noncooperatively with markedly different affinities (0.11 and 6.9 µM). The covalently attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity.

Our NMR data indicate that two Ca bind per recoverin molecule and that a global conformational change occurs when two Ca bind to the myristoylated protein. Many of the proton and N resonances of recoverin exhibit chemical shift changes when up to two Ca bind, but no marked changes were detected when more than two Ca equivalents were added. Sequence-specific assignments have been obtained for the backbone amide (Ames et al., 1994) and tryptophan indole resonances. (^2)These assignments identify the residues that undergo structural change upon Ca binding. Almost all backbone amide resonances from residues in both the amino- and carboxyl-terminal domains change chemical shift simultaneously when up to two Ca bind (Fig. 8). The Ca-induced conformational change also affects the side chain indole resonances of Trp-31, Trp-104, and Trp-156. These results suggest that Ca-induced structural changes are occurring throughout the protein in a concerted fashion.

From the amino acid sequence and x-ray structure it seems clear that only EF-2 and EF-3 can bind Ca. Which is the high affinity site? A large data base of amino acid sequences of the EF-hand protein family suggests that aspartic acid is highly conserved at the third position of the 12-residue Ca binding loop, whereas the fifth position can be more frequently substituted by other amino acids such as asparagine (Wylie and Vanaman, 1988). Sequence comparison of the Ca binding loops of EF-2 and EF-3 (Fig. 1) indicates that the most favorable residue (aspartic acid) at the third position is present in EF-3, whereas it is substituted with asparagine in EF-2. This suggests that EF-3 has a higher affinity for Ca than EF-2. Furthermore, the x-ray structure shows Ca bound to EF-3 and not EF-2 (Flaherty et al., 1993). Sequence-specific NMR assignments for ^1H resonances at 5.61 and -0.47 ppm (Fig. 6A), which monitor Ca binding to the high affinity site, will determine whether EF-3 is in fact the high affinity site.

The myristoyl group promotes cooperative binding of Ca to EF-2 and EF-3 sites. What is the structural basis of this cooperativity? The x-ray crystal structure of unmyristoylated recoverin with one Ca bound (Flaherty et al., 1993) showed that the helices of EF-3 pack closely against the amino-terminal helix of EF-2, resulting in a compact globular fold. In addition, the myristoyl group is thought to bind to a hydrophobic cleft located between EF-1 and EF-2. The present study indicates that Ca binding to EF-2 and EF-3 is independent when the myristoyl group is absent. The myristoyl group may, therefore, alter EF-2 to allow communication between EF-2 and EF-3. Specifically, the amino-terminal helix of EF-2 is unstable in the myristoylated protein (Ames et al., 1994), whereas it is stable in the unmyristoylated form (Flaherty et al., 1993).

The effect of the myristoyl group on the Ca binding cooperativity is somewhat analogous to the effect of a target peptide on Ca binding to calmodulin. In the absence of a target, there is little cooperativity between the amino- and carboxyl-terminal domains of calmodulin (Minowa and Yagi, 1984; Klee, 1988). In the presence of a target such as a 26-residue peptide derived from myosin light-chain kinase and mastoparan, the two domains interact with each other, resulting in highly cooperative Ca binding (Yazawa et al., 1987). This target-induced interaction between the two domains of calmodulin has been accounted for by the compact structures of calmodulin complexed with myosin light chain kinase peptides determined by NMR spectroscopy (Ikura et al., 1992) and x-ray crystallography (Meador et al., 1992). For recoverin, the myristoyl group may similarly induce an interaction between the amino- and carboxyl-terminal domains, perhaps by binding to the interface of the two domains. The crystal structure of Ca-bound unmyristoylated recoverin (Flaherty et al., 1993) shows a compact shape. Recent NMR studies (Ames et al., 1994) and small-angle x-ray scattering studies (Kataoka et al., 1993) on myristoylated recoverin also suggest a compact shape even in the absence of Ca. Hence, cooperative Ca binding by myristoylated recoverin may be the consequence of rather subtle structural differences brought about by the myristoyl group.

A concerted allosteric mechanism is proposed to explain how the myristoyl group induces cooperative Ca binding to recoverin. The model shown in Fig. 4is like the Monod-Changeux-Wyman model (Monod et al., 1965), except that the Ca binding sites are taken to be intrinsically different and hence have different affinities. The existence of two protein conformations T and R, defined by the Ca-myristoyl switch (Zozulya and Stryer, 1992), is supported by the fluorescence and NMR data presented in this study. The T state can be pictured to have the myristoyl group buried inside the protein core, allowing recoverin to be stable in solution. The R state places the myristoyl group outside so that it can interact with disc membranes or another protein. The intrinsic dissociation constants of the R state for Ca are K(1) and K(2), and those of the T state are K(1)/c and K(2)/c, where c is estimated to be less than 10. Thus, Ca binds at least 10,000-fold as tightly to R than to T. Unlike the Monod-Wyman-Changeux model, K(1) and K(2) are allowed to be different and are assumed to be 0.11 and 6.9 µM in the R state, the same as measured for unmyristoylated recoverin. Based on these values of K(1) and K(2), the ratio (L) of the unliganded form of T to that of R is estimated to be 400 for myristoylated and <0.05 for unmyristoylated recoverin. Using the obtained values of L and c (Table 1), myristoylated recoverin is calculated to be almost exclusively in the T state in the absence of Ca (99.8% T for L = 400). The binding of each Ca changes the equilibrium between T and R by a factor of c in the direction of R. When both sites are occupied, the ratio of T to R ([T]/[R]) is 4 times 10. Therefore, about 5 kcal/mol of the binding energy of each Ca is used to drive the conformational equilibrium to the R state so that recoverin is almost entirely in the R state when two Ca bind.

The apparent low Ca affinity of myristoylated recoverin in solution is linked to an unfavorable extrusion of the myristoyl group during the T to R transition (L = 400, DeltaG° = +3.5 kcal/mol). The T R transition is opposed in solution because the myristoyl group is then forced to interact with H(2)O. A membrane environment should stabilize the extruded myristoyl group in the R state and hence cause L to decrease. Fig. 2A shows the calculated effect of lowering L on the Ca binding curve. The apparent Ca affinity of myristoylated recoverin is brought into the physiological range when L is lowered. An L value of 40 predicts a binding curve with apparent dissociation constant of about 4 µM. This calculated curve is similar to the curve describing the binding of myristoylated recoverin to rod outer segment membranes in which half-maximal binding occurs at 4 µM Ca (Zozulya and Stryer, 1992). S-Modulin, a close relative of recoverin found in frog rods, was found to associate with frog rod outer segment membranes and inhibit phosphorylation of rhodopsin with a Ca dissociaton constant of 1 µM (Kawamura, 1993).

The allosteric model in Fig. 4has implications concerning the kinetics of the Ca myristoyl switch. Upper limits on the rate constants for binding and release of Ca from the R and T states can be inferred. For both states, the rate constant for binding Ca is limited by diffusion to be less than about 10^9M s. The dissociation rates from the high affinity (EF-3) and low affinity (EF-2) sites can be calculated from the dissociation constant for each site. The calculated dissociation rate of Ca from the low affinity site (EF-2) in the R state is less than 7000 s and that from the high affinity site (EF-3) in the R state is less than 100 s. Similarly, the dissociation rates of Ca from the high and low affinity sites in the T state are less than 10^6 s and 7 times 10^7 s, respectively. The rate constants for the allosteric transitions between R and T are not known. Stopped-flow kinetic measurements of the Ca-induced tryptophan fluorescence changes of myristoylated recoverin and of Ca uptake by fluorescent buffers are currently in progress to determine the kinetics of Ca binding and the allosteric transition.


FOOTNOTES

*
This work was supported in part by Grants from the National Institutes of Health (GM24032 and EY02005) (to L. S.) and by grants from the Medical Research Council of Canada and the Human Frontier Science Program Organization (to M. I.). 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 National Institutes of Health Postdoctoral Fellowship EY06418-02.

Supported by an Ontario Cancer Institute/Amgen postdoctoral fellowship.

**
Recipient of a Medical Research Council of Canada Scholarship.

§§
To whom correspondence may be addressed.

(^1)
The abbreviations used: 1D, one-dimensional; HSQC, heteronuclear single quantum correlation; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

(^2)
T. Tanaka, unpublished results.


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