©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Determinants of Ion Specificity on EF-hands Sites
CONVERSION OF THE Ca/Mg SITE OF SMOOTH MUSCLE MYOSIN REGULATORY LIGHT CHAIN INTO A Ca-SPECIFIC SITE (*)

(Received for publication, October 11, 1994; and in revised form, December 12, 1994)

A. C. R. da Silva (§) J. Kendrick-Jones (1) F. C. Reinach

From the Departamento de Bioquimica, Instituto de Química, Universidade de São Paulo, C.P. 20780 CEP 01498 São Paulo SP, Brazil Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Calcium binding proteins mediate a large number of cellular processes. These processes respond to micromolar fluctuations of cytosolic calcium in the presence of a large excess of magnesium. The metal binding sites present in these proteins are either calcium-specific (regulatory sites) or capable of binding both calcium and magnesium (structural sites). Using site-directed mutagenesis we were able to convert the single Ca/Mg site present in chicken smooth muscle myosin regulatory light chain (RLC) into a Ca-specific site. The replacement of the aspartic acid present in the 12th position (-Z coordinating position) of the metal binding loop with a glutamic acid increases calcium affinity and abolishes magnesium binding, rendering the site calcium-specific. To explain this observation, we hypothesize that restrictions on the ability of side chains to change conformation, contributing one (for Mg binding) or two (for Ca binding) coordinations could alter the metal specificity in EF-hands. Other mutations which decrease or abolish calcium binding have also been characterized. When used to substitute the endogenous scallop myosin RLC, these mutants were capable of restoring the Ca regulation to the actin-activated myosin ATPase demonstrating that in these hybrid myosins, the regulatory function of the Ca-specific site (present on the essential light chain) does not depend on the occupancy of the Ca/Mg site (present on the regulatory light chain).


INTRODUCTION

During muscle activation, intracellular calcium levels fluctuate between 0.1 and 10 µM, while magnesium concentrations are kept constant at much higher levels (2 mM). Regulatory proteins must therefore be capable of detecting these changes in calcium concentrations in the presence of a thousand-fold molar excess of magnesium. Here we investigate the molecular basis for this discrimination. The EF-hand Ca binding motif (1, 2, 3) is responsible for this discrimination in most regulatory proteins. It consists of a 12-amino acid binding loop flanked by two perpendicular alpha-helices (Fig. 1)(1) . Four functional copies of this motif are found in calmodulin and troponin-C, two in the parvalbumins and one in the myosin light chains(3, 4) . EF-hands are either calcium-specific (Ca sites) or capable of binding calcium and magnesium (Ca/Mg sites). Due to the high concentration of Mg in the cytoplasm, Ca/Mg sites are thought to be occupied by Mgin vivo and involved in stabilizing the structure of proteins while the Ca-specific sites perform the regulatory functions. The structural basis for divalent metal discrimination by these sites remains unknown despite the fact that the crystallographic structures of a number of EF-hand-containing proteins have been determined(3, 4, 5, 6) . Comparative studies suggest that Ca/Mg discrimination by EF-hand motifs relies on the radius differences between the ions (1.06 Å for Ca and 0.76 Å for Mg) and the consequent energy requirements for the replacement of the hydration sphere with protein ligands(7, 8) .


Figure 1: A, the structure of an EF-hand calcium binding site showing the coordination of the metal ion. The EF-hand calcium binding motif is composed of a loop flanked by two helices. The helix to the NH(2)-terminal side of the binding loop is shown coming up, perpendicular to the plane of the picture. The loop containing the coordinating side chains encircles the calcium ion (pink) in the plane of the picture and the helix which emerges from the COOH-terminal side of the loop exits to the right. The oxygens that coordinate the ion directly are shown in red, the oxygen from the water molecule in white, and the oxygen from the side chain that coordinates the water molecule in yellow. The first coordinating side chain (X; position 1 in the loop) is hidden under the calcium ion, below the plane of the figure. The second (Y; position 3) and third (Z; position 5) coordinating side chains can be observed as the main chain goes around the calcium ion. The fourth (-Y; position 7) coordination is provided by a main chain oxygen and is hidden behind the metal ion. The fifth (-X; position 9) coordination (yellow) is indirect and occurs by way of a water molecule (white). The sixth and seventh (-Z; position 12) are provided by a bidentate glutamate which reaches in from the first turn of the COOH-terminal helix and contributes two oxygens. The five coordinating oxygens from positions 3, 5, 7, and 12 form a pentagon in the plane of the figure while the oxygens from position 1 and from the water molecule form the vertices of the two pyramids originating from the pentagon. The figure was constructed with the coordinates from site III of troponin-C (19) (5TNC, Brookhaven Data Bank). B, location of the mutants in the sequence of the binding loop. The sequence of the cation binding loop of site III of troponin-C (shown above) and that of site I of smooth muscle RLC are aligned. Also shown are the metal ion coordination positions (X, Y, Z etc.) and the numbering used for the residues along the loop. The mutations constructed are shown below the smooth muscle RLC sequence.



The chicken smooth muscle myosin regulatory light chain (RLC) (9) has a combination of characteristics which would be desirable in a model system in which we can investigate the determinants of ion specificity. It contains a single Ca/Mg site which retains high affinity for metals when removed from myosin, a property not found in the skeletal or scallop RLCs(10, 11, 12) . It restores calcium regulation to desensitized scallop myosin, allowing us to analyze the effects of the mutations on the regulation of the actomyosin ATPase activity(13) . And finally, although the crystallographic structure of the smooth muscle RLC (^1)has not been determined, that of skeletal muscle (14) and the scallop muscle (4) have been elucidated.

Since the crystal structure of the smooth muscle RLC is not known, we compared EF-hand calcium binding loops from proteins with known crystal structures and ion specificity in order to select the positions along the loop most likely to be involved in Ca/Mg discrimination(3, 15, 16) . The 4th position in the loop is occupied by a glycine residue in over 50% of the known loops and in 100% of the Ca-specific loops, suggesting that the extra flexibility and/or small side chain provided by this glycine might be important for cation discrimination. In the RLC, this position is occupied by an arginine. The bound cation can have all its water molecules substituted with protein ligands, as in the case of the Ca/Mg site of parvalbumin (17, 18) , or keep a single water molecule, as in the case of the Ca/Mg sites of troponin C (19) and the Ca-specific sites of calmodulin(20) . It is the amino acid side chain at position 9 of the calcium binding loop which determines if a water molecule remains associated with the bound cation (Fig. 1). If a long side chain is present, for example glutamic acid in parvalbumin, it coordinates the ion directly. Shorter side chains, for example serine or aspartic acid, allow for the presence of a water molecule that is sometimes hydrogen-bonded to the side chain. The nature of the residue in this 9th position may influence the size of the coordination sphere and therefore be involved in ion discrimination. It has also been recently demonstrated that the residue present in this position determines the ``on'' and ``off'' rates of metal binding to the site(21) . In oncomodulin it has been demonstrated that the substitution of a glutamic acid for an aspartic acid in this position has little effect on the specificity of this site(22) . The 12th position (-Z coordination) is occupied by a glutamic acid (85%) or aspartic acid (10%) in most loops irrespective of ion specificity (5, 6, 15, 16, 23) (Fig. 1). It has been observed in the structure of the sarcoplasmic Ca binding protein that the loop containing an aspartic acid at the 12th position is smaller and more compact when compared with loops containing glutamic acid at the same position(5) . Strynadka and James (3) proposed that the acidic residue at position 12 plays a central role in adapting the loop of Ca/Mg sites when different cations are bound. The two oxygens from its carboxylate group could either coordinate the Ca in a bidentate manner or change its conformation, providing a single coordination for Mg. This conformational change has been observed in the structure of pike parvalbumin crystallized with either Ca or Mg in its second site(18) . As RLC possess an aspartic acid at position 12, the site probably resembles the structure of sarcoplasmic Ca binding protein and we would expect that the substitution of aspartic acid for glutamic acid could open the site and favor the binding of larger ions.

Site-directed mutants were constructed to tests these hypotheses. We found that the replacement of the aspartic acid present in the 12th position with a glutamic acid renders the site calcium-specific. Other mutants increased, decreased, or abolished calcium affinity without drastically altering the specificity. All mutants were used to replace the endogenous RLC from scallop muscle. Since the mutants restored calcium regulation and the calcium-specific site present in the essential light chain, we conclude that in these hybrids the occupancy of the RLC site is not essential for proper regulation of the myosin ATPase.


MATERIALS AND METHODS

Chemical Reagents

All reagents were analytical grade. Milli-Q deionized water (Millipore Corp.) was used in all experiments.

Construction, Expression, and Purification of Mutant RLCs

A cDNA encoding the smooth muscle regulatory light chain (9) was cloned in M13mp19. A clone containing the 5`-end of the mRNA facing the universal primer was selected for mutagenesis. Oligonucleotide mutagenesis was performed as described (24) using the following oligonucleotides: D9S, 5`TGGGTTCATTTCCAAGGAGGATC; D12A, 5`ACAAGGAGGCTCTGCATGACA; R4G, 5`CGACCAGAACGGTGACGGGTTCA; D12E, 5`ACAAGGAGGAGCTGCATGACA; and D9E, 5`GCTTCATTGAGAAGGAGGATC.

The numbers refer to the position of the mutated amino acid in the EF-hand site. The double mutant R4G/D12E was generated by performing the second mutation D12E on the R4G mutant template. The mutated coding region of the gene was sequenced and used to replace the wild type fragment of the cDNA between the single HindIII/EcoRI sites in pMW172(25) . The presence of the mutations was confirmed by sequencing the final expression vector. Expression of smooth RLC was performed as described(25) . The bacterial pellet was resuspended in 25% sucrose, 2 mM EDTA, 2 mM EGTA, 50 mM Tris-Cl, pH 8.0, and lysed in a French Press (16,000 p.s.i.). After lysis, trichloroacetic acid was added to a final concentration of 3%, and the precipitated protein was resuspended in 25 mM Tris-Cl, pH 7.5, 0.5 mM MgCl(2), 2 mM beta-mercaptoethanol. The pH was adjusted to 7.0 with Tris base, and the suspension was dialyzed overnight against the same buffer. After centrifugation, 6 M urea was added to the supernatant. The pellet was resuspended in 6 M urea in the same buffer.

The solutions were clarified by centrifugation and separately applied to a DEAE-Sepharose FF equilibrated with 6 M urea, 25 mM Tris-Cl, pH 7.5, 0.5 mM MgCl(2), 2 mM beta-mercaptoethanol. The pure proteins were eluted with a 0-400 mM NaCl gradient. The final purification was achieved on Phenyl-Sepharose 6B equilibrated in 4 M NaCl, 25 mM Tris-Cl, pH 7.5, 0.5 mM MgCl(2) using a gradient from 4.0 to 0 M NaCl.

Direct Ca Binding Measurements

Calcium was removed from the RLCs and from the RLC/heavy chain hybrids as described (26) . Ca binding was measured by flow dialysis as described previously (26) with some modifications. The assay solution contained: 50 mM NaCl, 20 mM imidazole, pH 7.0, 2 mM beta-mercaptoethanol. Protein concentration in the assay was 65 µM (molecular mass of RLC is 19.7 kDa). Calcium contamination in the buffers was determined to be below 10M by atomic absorption spectroscopy. The concentrations of CaCl(2) stock solutions were 5 mM and 40 mM, and aliquots of 5 to 25 µl were added every fifth fraction. The final addition of CaCl(2) (50 µl of a 1 M stock) was at fraction 55. Experiments were performed in the absence and presence of 2 mM Mg. Three data sets under each condition were fitted using a nonlinear regression curve-fitting procedure to :

where y is the number of sites filled at each [Ca] and Kis the apparent dissociation constant. Since saturation occurred at values higher than 1 mol of Ca/mol of protein, a correction factor (n`) was determined during the fitting procedure to account for nonspecific binding. Nonspecific Ca binding to the surface of the dialysis membrane and the vessel can be demonstrated in the absence of protein. The ability of magnesium to compete off the bound calcium was determined as follows. RLC was added to the upper chamber at the same concentration used in the Ca measurements, in the same buffer. At the tenth fraction, the calcium concentration was increased by 40 µM, and aliquots of 2 to 40 µl of 250 mM MgCl(2) were added every fifth fraction, resulting in a final concentration of 10 mM after the ninth addition. At the end of the titration, CaCl(2) was added to a final concentration of 10 mM. The fourth and fifth fractions after each addition were used to determine the amount of calcium bound. K was determined by fitting three data sets to , using a nonlinear regression curve-fitting procedure.

where y` is the number of sites filled at each [Mg] concentration, K and K are the apparent dissociation constants, and n` a correction factor to nonspecific binding. K was obtained from .

Reconstitution of DMF with Mutant RLCs and Measurements of Actin-activated Myosin ATPase

Desensitized scallop myofibril (DMF) and DMF reconstituted with RLC were prepared as described previously(10, 27) . The actin-activated myosin ATPase activities were measured using a pH-stat as described (10) with the following modification. The activities were measured on 2 separate aliquots of the same sample, one in the presence of 0.1 mM EGTA and other in the presence of 0.1 mM EGTA and 0.12 mM CaCl(2). To determine the binding of RLC to DMF, aliquots of the DMF-RLC hybrids were spun in a microcentrifuge tube, resuspended in SDS-polyacrylamide gel electrophoresis or urea/glycerol sample buffer, and run on SDS or glycerol/urea gels(10) . To determine the molar ratios of the two light chains, the gels were analyzed as described previously(10) . The molar ratios of RLC/ELC was determined to be 0.65 ± 0.2 (D12A), 0.9 ± 0.2 (D9S), 0.75 ± 0.2 (R4G), 0.70 ± 0.2 (D12E), 0.77 ± 0.2 (D9E), 0.55 ± 0.2 (R4G/D12E), 0.85 ± 0.1 (scallop wild type), and 0.71 ± 0.2 (smooth wild type).

Reconstitution of Desensitized Myosin with RLC

Desensitized myosin was prepared as described in(28) . To reconstitute the myosin, aliquots of desensitized myosin (10 mg) were incubated with RLC (1.3 mg) in 0.6 M NaCl at pH 7.0 for 1 h on ice. After incubation, the myosin solution was dialyzed against 50 mM NaCl, 20 mM imidazole, pH 7.0, 1 mM MgCl(2), 0.1 mM dithiothreitol, to precipitate the myosin. The myosin filaments were washed in 10 ml of buffer A (50 mM NaCl, 20 mM imidazole, pH 7.0, 2 mM beta-mercaptoethanol) twice, buffer A with 2 mM EDTA three times, and finally in buffer A six times to remove the EDTA. The myosin was resuspended in 6 ml of buffer A and split in 2 aliquots. The first (1 ml) was used to measure the protein concentration and its RLC content by analysis on urea/glycerol gel. The other aliquot (5 ml) was used to measure Ca binding by flow dialysis.


RESULTS

Calcium Binding Properties of Mutant Chicken Smooth Muscle Myosin RLCs

To study the determinants of specificity on the EF-hand metal binding sites, we produced six chicken smooth muscle myosin RLCs containing point mutations on the single Ca/Mg EF-hand site. Mutant R4G, in which the arginine in the 4th position of the calcium binding loop was changed for glycine, was constructed in an attempt to increase the flexibility of the loop. D9S and D9E were constructed in order to test if the length of the side chain would influence the specificity of the site by altering the coordination of the water molecule that remains bound to the metal ion. D12E and the double mutant R4G/D12E were designed to test the effect of the length of the bidentate coordinating residue on cation binding specificity. Finally, D12A was designed as a negative control since this mutant has been shown to abolish metal binding in both the skeletal muscle (11) and scallop muscle (12) RLCs. These proteins were expressed in Escherichia coli, purified to homogeneity, and used to determine calcium binding by flow dialysis.

As expected for a single Ca/Mg binding site, wild type RLC binds approximately 1 mol of Ca per mol of RLC (Fig. 2A). In the presence of magnesium, this ability is almost abolished (Fig. 2B). The substitution of the arginine in position 4 of the loop with a glycine (R4G, Fig. 1) was a silent mutation. As the wild type RLC, it binds calcium with an affinity close to 10 µM (Fig. 2A; Table 1), and its affinity for calcium is reduced 9-fold (to 90 µM) in the presence of 2 mM Mg (Fig. 2B; Table 1). The affinity of wild type RLC and mutant R4G for magnesium is close to 400 µM (Fig. 2C; Table 1).


Figure 2: Calcium binding to isolated recombinant smooth muscle RLC (bullet) and mutants: D9S (down triangle), D12A (box), R4G (up triangle), D12E (), D9E (), and R4G/D12E (). Ca binding was measured as a function of Ca concentration in the absence (A) and presence (B) of 2 mM Mg. In C, the capacity of Mg to displace bound Ca was measured as a function of Mg concentration. The data points represent the mean from three independent assays. Standard deviations of the mean are shown as bars (n = 3). The lines are curve-fitted according to the parameters shown in Table 1.





In the second set of mutants, we changed the length of the side chain in position 9, replacing the aspartic acid by either a glutamic or a serine residue (D9E and D9S, Fig. 1). When compared with the wild type RLC, these mutants showed a 2-fold reduction in calcium affinity both in the presence and absence of magnesium (Fig. 2, A and B; Table 1) and also a reduction in magnesium affinity (Table 1). This decrease in affinity did not result in a significant change in ion selectivity as defined by the ratio K/K (Table 1).

A dramatic increase in ion selectivity was obtained when the side chain of position 12 was made longer by replacing the aspartic with a glutamic acid (D12E and R4G/D12E, Fig. 1). When compared with wild type RLC, we observed in both mutants a 2-fold increase in affinity for calcium in the absence of magnesium (Fig. 2A) and a 100-fold increase in affinity in the presence of 2 mM Mg (Fig. 2B; Table 1). This rendered the site calcium-specific, and bound calcium could not be displaced by up to 10 mM magnesium (Fig. 2C). The lower saturation values observed for mutants D12E and D12E/R4G in the presence of Mg (Fig. 2, A and B) are probably related to a reduction in nonspecific Ca binding (Table 1).

These results demonstrate that the Ca/Mg binding site present in the isolated protein was transformed into a Ca-specific site.

Regulatory Properties of Mutant RLCs

The mutant RLCs were incorporated into scallop-desensitized myofibril in order to check their ability to restore Ca regulation to the ATPase activity. Removal of the endogenous RLC from scallop myosin abolishes calcium regulation which is restored when scallop or wild type chicken smooth muscle RLCs are added back(29) . We observed that, regardless of the properties of their metal binding site, all our smooth muscle RLC mutants were capable of forming stable complexes with the scallop myosin heavy chain (Fig. 3) and restoring Ca regulation to the scallop actomyosin ATPase (Table 2). The observation that mutant D12E, with a calcium-specific site, was able to inhibit the ATPase in the absence of Ca suggested that the smooth muscle RLC behaves differently from the skeletal and scallop RLCs which need an intact Ca/Mg binding site to regulate the ATPase activity(11, 12) . This was confirmed using mutant D12A which has its metal binding largely abolished (Table 1; Fig. 2) but is still capable of regulating the ATPase activity of scallop myosin (Table 2). The same mutation when performed on the skeletal RLC (11) or scallop RLC (12) locked the ATPase in the on state, irrespective of the calcium concentration, a result that lead to the suggestion that this site might be involved in the inhibition of the scallop actomyosin ATPase. To exclude the possibility that the Ca/Mg site was restored in the D12A mutant when the RLC was incorporated into scallop myosin, the calcium binding properties of myosin reconstituted with mutant D12A were measured. While the desensitized myosin (containing the heavy chain and the essential light chain) showed only background calcium binding (less than 1 mol of Ca/mol of myosin head), the incorporation of wild type smooth muscle RLC restored the two calcium binding sites to the head, i.e. a Ca/Mg site present in the RLC and the Ca-specific site made up by the ELC and the scallop myosin heavy chain (Fig. 4)(4, 30) . Mutant D12A was able to restore only one metal binding site, most likely the Ca-specific site responsible for regulation(4, 30, 31) (Fig. 4). These results clearly demonstrate that as long as the RLC is able to restore the Ca-specific site on the scallop ELC, regulation is achieved irrespective of the occupancy of the RLC Ca/Mg site.


Figure 3: Reconstitution of desensitized scallop myofibrils with recombinant and smooth RLCs. A, SDS-polyacrylamide gel electrophoresis (15%); B, urea-glycerol gel of desensitized scallop myofibril (DMF) reconstituted with the following regulatory light chain: 1, none; 2, smooth wild type; 3, smooth D12A; 4, scallop RLC; 5, smooth D9S; 6, smooth R4G; 7, smooth D12E; 8, smooth D9E; 9, smooth R4G/D12E. The arrows indicate the regulatory light chain. Note that in SDS gels the scallop RLC co-migrates with the ELC.






Figure 4: Ca binding properties of desensitized scallop myosin reconstituted with recombinant smooth RLCs. A, Ca binding to scallop myosin as a function of Ca concentration. Desensitized myosin () was reconstituted with wild type smooth RLC (bullet) or smooth RLC D12A (). The data points represent the mean from three independent assays. B, urea/glycerol polyacrylamide gel electrophoresis of desensitized myosin alone (1) and reconstituted with wild type smooth muscle RLC (2) and D12A (3). The arrow indicates the RLC.




DISCUSSION

Metal Specificity of EF-hands

Our results demonstrate that the replacement of the aspartic acid present in the 12th coordinating position with a glutamic acid converts the single Ca/Mg site present in the isolated chicken smooth muscle RLC into a Ca-specific site. A definite structural interpretation for this result is difficult at this time because the crystal structure of smooth muscle RLCs has not been determined. We can only infer how the coordination of the ions is organized in these proteins. This inference could be based on the crystal structures of the two regulatory myosin light chains already determined (from scallop (4) and skeletal (14) muscle myosins). However, in both cases, the structure that was determined was that of the RLC/myosin heavy chain complex and not of the isolated RLC. It should also be pointed out that the presence of a glutamic acid residue in position 12 is not sufficient by itself to confer calcium specificity to every EF-hand since many well characterized Ca/Mg EF-hand sites have a glutamic residue in the 12th position(15) . Given these limitations, we would like to put forward a possible interpretation that may explain this increase in specificity.

The fact that the increase in calcium selectivity was obtained without a decrease in calcium affinity (Table 1) suggests that the structural modification caused by the mutation does not affect the Ca binding but drastically reduces Mg binding. Since Mg is smaller than Ca, we would intuitively expect that an increase in the length of the side chain in the 12th position should favor the binding of the smaller ion, contrary to what we observed. Indeed, our results with the mutants in the 9th coordinating position where we both increased (D9E) or decreased (D9S) the length of the side chain without detecting changes in cation specificity support the idea that the specificity is not determined directly by the length of the coordinating side chain. In pike parvalbumin, where the crystal structure of both the Ca and Mg forms have been determined(18) , it was found that upon Mg binding the major change in the loop to accommodate the smaller ion was a rotation of the side chain of the residue in the 12th coordinating position (-X position). In the presence of Ca, the two carboxylate oxygens of this side chain form part of seven coordinating oxygens organized into a bi-pentagonal pyramid. In the presence of Mg, this side chain rotates so that it only contributes one ligand to the six-membered octahedral coordination shell. This type of rotation has been postulated as the conformational change necessary to accommodate the smaller Mg ion(3) .

We would like to propose that restrictions on the rotation of coordinating side chains contribute to the determination of the ion specificity of EF-hands. Accordingly, in the wild type smooth muscle RLC, the aspartate side chain in the 12th position could rotate and accommodate either Mg or Ca. In the mutant protein, this side chain would have its rotation sterically blocked into the bidentate configuration. The site would not be able to accommodate the smaller Mg ion, thereby reducing its affinity for this ion. Furthermore, if the longer glutamic side chain is locked in the bidentate orientation, preferred by the Ca ion, and cannot rotate to provide the single coordination necessary to accommodate the Mg ion, it is expected that the affinity for Mg would be greatly reduced. This restriction in rotation should also favor calcium binding, increasing the affinity for this ion, as we observed in the D12E mutant. Rotation of side chains, although predicted (3) and observed before(18) , have not been postulated as a possible explanation for ion selectivity. Direct evaluation of this hypothesis must await the determination of the structure of the smooth muscle RLC.

This idea could also explain why it has been impossible to predict the ion specificity of EF-hands based only in the primary sequence. Because the rotation is limited by the local environment of the side chain, it would be difficult, if not impossible, to predict from the primary structure if a given side chain is able to rotate in a particular environment.

Regulation of the ATPase

In scallop muscle, the actomyosin MgATPase is activated when calcium binds to a Ca-specific binding site present in the essential light chain (ELC)(4) . The presence of this Ca-specific site depends on the interaction of the ELC with the heavy chain and the RLC. When the RLC is removed, this site is lost even though the ELC remains bound to the myosin head. The elucidation of the structure of the regulatory domain of this myosin (4) clarified how the COOH-terminal half of the RLC contributes toward the structure of the ELC Ca-specific site. It has been demonstrated that mutations that destroy the Ca/Mg site present in the RLC lock the ATPase in the on state, suggesting that this site is important for the inhibition of the ATPase(11, 12) . It has also been demonstrated that wild type smooth muscle RLC can restore calcium regulation to the scallop myosin(25) .

When used to replace the endogenous RLC in scallop myosin, our mutant smooth muscle RLCs were capable of restoring calcium regulation to the system. The observation that the calcium-specific mutant (D12E) was able to inhibit in the absence of calcium (a situation where the site should be empty) suggested that the occupancy of the RLC site is not necessary for inhibition. This difference in behavior when compared with the results obtained with the skeletal or scallop system(11, 12) was confirmed when we tested a smooth muscle RLC with the Ca/Mg site destroyed (D12A). To exclude the possibility that the D12A mutant of smooth muscle RLC regained its metal binding site when incorporated into the scallop myosin, we measured calcium binding to the reconstituted myosins. These measurements demonstrated that this mutant is capable of restoring the calcium-specific, regulatory site as expected from its ability to restore the regulation of the ATPase.

These results demonstrate that it is possible to obtain hybrids between scallop myosin and smooth muscle RLCs with a single Ca site still capable of being regulated by calcium. This strongly supports the idea that the RLC Ca/Mg is not necessary for the reconstitution of the Ca-specific site on the ELC or for the regulation of the ATPase. Rather, the RLC Ca/Mg site probably plays a structural role, as expected from its specificity. It remains to be determined why it has not been possible to obtain similar results with hybrids constructed using mutants of the endogenous or skeletal muscle RLCs.


FOOTNOTES

*
This work was supported in part by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo, Programa de Apoio ao Desenvolvimento Cientifico e Technologico (PADCT), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, and the Rockefeller Foundation. 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 Fundação de Amparo à Pesquisa do Estado de São Paulo.

(^1)
The abbreviations used are: RLC, regulatory light chain; DMF, desensitized scallop myofibril; ELC, essential light chain.


ACKNOWLEDGEMENTS

We thank C. Cohen for providing us with the coordinates of the scallop myosin light chain before publication.


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