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Originally published In Press as doi:10.1074/jbc.M310085200 on November 25, 2003

J. Biol. Chem., Vol. 279, Issue 7, 5380-5386, February 13, 2004
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Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum*

Chris Ryan, David L. Stokes{ddagger}, Minggui Chen§, Zhimin Zhang, and Peter M. D. Hardwicke

From the Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901

Received for publication, September 10, 2003 , and in revised form, November 12, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca2+-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca2+-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sarco(endo)plasmic reticulum ATPases (SERCAs)1 transport Ca2+ against an electrochemical gradient from the cytoplasm into the intracellular membranous compartment of the sarcoplasmic or endoplasmic reticulum and play a major role in Ca2+ homeostasis in both muscle and non-muscle cells (1, 2). In the course of transporting Ca2+, these enzymes pass through a number of relatively well defined intermediate biochemical states that can be isolated and studied. These include forms in which the {beta}-carboxyl group of a specific aspartyl residue (Asp351 in the case of rabbit SERCA1a) is present as an acylphosphate mixed anhydride. Much of the experimental data has been interpreted in terms of the enzyme being able to exist in two basic forms: the E1 state, in which the enzyme possesses high affinity binding sites for Ca2+ and can be phosphorylated by ATP but not by Pi, and the E2 state, which possesses low affinity Ca2+ sites and can be phosphorylated by Pi but not by ATP (3).

Comparison of the tryptic digestion patterns of rabbit SERCA1a in its Ca2+-bound and -free forms has provided some of the strongest direct evidence supporting such a model where the enzyme can exist in two fundamentally different conformational states (4–6). Recent studies have found that the E1 (Ca2+)2 form of rabbit SERCa1a shows clear structural differences to the enzyme in its vanadate-stabilized Ca2+-free (E2) state (7–10). In the E1 form, the Actuator (A) domain is isolated from the nucleotide (N) and phosphorylation (P) domains, whereas in the E2 form all three domains are clustered together.

The Ca-ATPase from the cross-striated part of the adductor muscle of the sea scallop has been the subject of a number of biochemical and structural studies (11, 12). Provided the E2 form of the scallop Ca-ATPase is stabilized against loss of activity, it adopts a dimeric type of quaternary organization (13) that is absent in the E1 form of the enzyme (14), and in both the E1~P and E2-P states the enzyme is arranged in parallel instead of antiparallel helical strands in the tubular vesicles with only a single asymmetric subunit in each unit cell (p1 lattice) (15).

In the work reported here, tryptic digests of scallop SERCA detected differences in conformation between the E1 and E2 forms of the enzyme associated with the N and P subdomains as well as with the A domain. It was found that some features of the proteolytic stability normally associated with the E2 state were also observed when ligands ranging from AMP-PNP to simple orthophosphate were bound to the catalytic site of the unphosphorylated E1 form of the enzyme, despite saturation of the Ca2+-binding sites. Thus, occupation of the catalytic site on E1 could induce structural changes that resembled in some respects those produced by emptying of the high affinity Ca2+ sites; however, this modified structural form of the E1 state of the scallop Ca-ATPase was unique and differed from E2. The effect of covalent phosphorylation of the scallop Ca-ATPase was also investigated, when the E2-P form was found to be exceptionally stable, both in terms of enzymatic activity and resistance to proteolysis. In contrast, the E1~P form was highly susceptible to trypsin with a digestion pattern resembling that of the E1 (Ca2+)2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Deep-sea scallops (Placopecten magellanicus) were obtained from the Marine Biology Laboratory, Woods Hole, MA.

Preparation of Scallop FSR—SR vesicles were made from the crossstriated part of the adductor muscle as described previously (16, 17). One minor modification to the procedure was the removal of glycogen granules from the final preparation. Glycogen and its associated proteins are very common contaminants of SR vesicle preparations from rabbit skeletal muscle (18), and the same is true of scallop muscle membranes. In the case of the scallop SR, it was found that a simple and effective method to eliminate much of the glycogen was to layer the purified membrane fraction suspended in 0.32 M sucrose, 0.1 M KCl, 1 mM CaCl2,20mM MOPSNa, pH 7.0, onto 1.5 M sucrose, 0.1 M KCl, 1 mM CaCl2, 20 mM MOPSNa, pH 7.0, and centrifuge the preparation for 3 h at 1.5 x 105 x g. The glycogen particles collected as a clear button-like pellet at the bottom of the centrifuge tube, whereas the membranes, now largely free of glycogen, banded at the 0.32–1.5 M sucrose interface. Preparation of DOC-extracted scallop FSR was done as described previously (16).

Tryptic Digests—DOC-extracted scallop FSR was suspended at 1 mg ml–1 in 20% v/v ethylene glycol (Pierce), 0.15 M KCl, 50 mM MOPSNa, pH 7.0. The E1 pattern of tryptic fragments was obtained when the free Ca2+ concentration was above ~10 µM, but typically a total of 1 or 3 mM CaCl2 was present for digests in the E1 state. For digests of the E2 form, 10 mM EGTANa replaced CaCl2. Digestion was at room temperature with TPCK-treated trypsin (12,000 units/mg, dissolved in 1 mM HCl; Sigma) added in a 1:30 w/w ratio to SR protein (giving 400 units of activity/mg SR protein). Digestions were usually terminated by addition of AEBSF (Calbiochem) to a final concentration of 20 mM, followed by transfer of the sample to ice. The simultaneous addition of HCl to 1 mM with the 20 mM AEBSF proved a very effective way of stopping the digestion. The samples were centrifuged at 16,000 x g for 1/2 h at 4 °C and the supernatant removed. The pellets containing the membrane-bound products were washed to remove trapped protease by resuspension in 0.32 M sucrose, 1 mM AEBSF, 1 mM CaCl2 (E1 digests) or 1 mM EGTA (E2 digests), 25 mM MOPSNa, pH 7.0, followed by recentrifugation. After repeating the washing step, the trypsin-free samples were finally resuspended in 0.32 M sucrose, 1 mM CaCl2 (E1 digests), or 1 mM EGTA (E2 digests), 25 mM MOPSNa, pH 7.0, before addition of an equal volume of 2x Laemmli or Tricine sample buffer.

Electrophoresis—The discontinuous Tris-glycine and Tris-Tricine systems (19, 20) were used for running SDS-polyacrylamide gels. Sodium thioglycholate (0.1 mM) was present in the sample denaturation medium and cathode buffer.

Electroblotting and N-terminal Sequencing—SDS gels were blotted onto Immobilon-PSQ polyvinylidene difluoride in a medium of 10% v/v MeOH, 10 mM CAPSNa, pH 11, at 4 °C with a Bio-Rad Trans Blot apparatus. Blots were stained with 0.02% w/v Coomassie Brilliant Blue R.250 in 1 mM HCl, 50% v/v MeOH. Bands of interest were sent to the University of Florida for N-terminal sequencing.

Phosphorylation with PI—For digestions in the E2-P state, DOC-extracted scallop FSR was phosphorylated with Pi essentially as described previously (15, 21) at room temperature for 15 min before addition of trypsin.

Detection of E2-P Using TNP-ADP Superfluorescence—This was carried out using an SLM 8000c spectrofluorimeter thermostated at 25 °C. TNP-ADP (Molecular Probes) was added to 35 µg/ml–1 DOC-extracted scallop FSR suspended in 20% v/v glycerol, 15 mM EGTA-Tris, 15 mM MgCl2, 50 mM Mes-Tris, pH 6.0, to a final concentration of 2 µM. Phosphorylation was typically induced by addition of Pi in the form of H3PO4-Tris to 8.5 mM. Steady-state fluorescence measurements using 4-nm slits were carried out with excitation at 403 nm, and emission followed at 532 nm.

Enzyme Assays—The dependence of the Ca2+-activated ATPase activity of the DOC-extracted scallop FSR on ATPMg2– concentration was determined using a coupled enzyme assay as previously described (16), except that 5 mM Mg2+ was present (as MgCl2) in excess of the ATPMg2– concentration, according to Neet and Green (22). Assays were carried out in a Perkin-Elmer Lambda 40 spectrophotometer in a volume of 1 ml. The cell was thermostated at 25 °C.

Protein Concentration—The bicinchoninic acid method (23) was used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
General Observations—As previously described, on SDS gels the undigested DOC-extracted scallop FSR fraction showed essentially a single band of ~110 kDa known to contain the polypeptides of the Ca-ATPase (24) and a Na-Ca exchanger (25), the latter probably of sarcolemmal origin. Based on phosphorylation levels of the scallop SR obtained with both ATP and Pi (15, 21) and on its specific Ca2+-activated ATPase activity (13, 16), the Ca-ATPase must represent at least 90% of the 110-kDa material. None of the membrane-bound proteolytic fragments described in this report was derived from the Na-Ca exchanger, although soluble tryptic peptides originating in the exchanger have been identified (25). Traces of 2–3 peptides of 28–32 kDa that arose from contamination of the SR by other elements of the sarcolemma (possibly gap junctions) were sometimes present (15, 25, 26). Gels of tryptic digests where the scallop Ca-ATPase was in the E2 state (+ EGTA) showed two strong bands of apparent molecular mass ~56 and ~47 kDa, together with much weaker bands corresponding to peptides of ~88, ~37, and 22–25 kDa (Fig. 1A). Digestions in the E1 state (10 µM free Ca2+ or above) gave a pattern in which both the 56- and 47-kDa bands were less intense than in E2 digests (Fig. 1B), whereas the 88-, 37-, and 22–25-kDa bands were significantly stronger.



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  		FIG. 1.
Time courses for digestion of the scallop Ca-ATPase in its E1 and E2 states. Digestions were carried out as described under "Experimental Procedures." Proteolysis was halted by addition of AEBSF to 20 mM and placing the sample on ice. Digested samples were washed free of trypsin. A, time course under E1 conditions. Lane 1, markers; lane 2, undigested FSR control; lanes 3–8, membrane-bound fragments from tryptic digestion of 20 µg of scallop FSR at 0, 1, 3, 5, 10, and 30 min, respectively. B, time course under E2 conditions. Lane 1, markers; lane 2, undigested control; lanes 3–8, membrane-bound fragments from tryptic digestion of 20 µg of scallop FSR at 0, 1, 3, 5, 10, and 30 min, respectively.

 
Attempts to sequence the 110- and 56-kDa tryptic fragments by the Edman method suggested that they both had blocked N termini. Autoradiography of SDS gels of membranes that had been first proteolyzed in the E2 state and then phosphorylated with [{gamma}-32P]ATP showed labeling of the 56-kDa band with 32P as well as the intact ATPase (not shown). Rabbit SERCA1a has a blocked (acetylated) N terminus, as of course does its A tryptic fragment, and the tryptic A fragment representing the N-terminal half of SERCA1a contains Asp351. Thus, the 56-kDa peptide formed in scallop tryptic digests contained Asp350 and corresponded as expected to the scallop A tryptic peptide.2 The polypeptide with the apparent size of 47 kDa formed in the same (E2) digests was found by Edman N-terminal sequencing of its polyvinylidene difluoride blot to be produced by cleavage at Lys504-Val505 (scallop sequence, Ref. 27) in the N domain and thus represented the scallop B tryptic fragment.

Under otherwise identical conditions, a more rapid overall digestion process occurred in the E1 compared with the E2 state. Comparison of time courses obtained when the scallop Ca-ATPase was in its E1 and E2 forms (Fig. 1, A and B) showed that in the E1 state the A band was lost much more rapidly than in E2. Although the B fragment was more stable than the A fragment in digests carried out in the presence of Ca2+ (when the enzyme was in its E1 form), it was nevertheless broken down faster than in the presence of EGTA (when the enzyme was in its E2 form). Therefore, Ca2+-dependent cleavages occurred in both the N- and C-terminal halves of the scallop Ca-ATPase.

N-terminal sequencing showed that the 88-kDa fragment, which was formed in larger amounts in the E1 than the E2 state, arose by cleavage at Arg197-Ala198 (scallop sequence, Ref. 27) in the A domain. As with rabbit SERCA1a, this will be designated the T2 cleavage site. Therefore, for a subpopulation of the scallop Ca-ATPase molecules in the E1 state, the primary tryptic cleavage site was T2 rather than T1. The 37-kDa fragment was relatively stable under E1 conditions and was often observed to be a very prominent band on SDS gels of tryptic digests made with the E1 form of the scallop Ca-ATPase (Fig. 1A). N-terminal sequencing showed that this peptide arose by cleavage at the Lys582-Phe583 peptide bond (scallop sequence, Ref. 27) in the N domain, which will be designated as the T3 site in the scallop enzyme. N-terminal sequencing of the 22 and 24-kDa peptides preferentially formed in the E1 state showed that formation of both of these peptides involved cleavage of the Lys727-Ser728 bond in the C-terminal component of the P domain. The Lys727-Ser728 bond will be designated as the T4 tryptic cleavage site in the scallop Ca-ATPase.

Effect of AMP-PNP, AMP-PCP, and ADP on the E1 Form of Scallop SR Ca-ATPase—When AMP-PNP was present at concentrations at or above ~0.1 mM in tryptic digests of the scallop Ca-ATPase in its E1 state (with free Ca2+ > 10 µM), substantial stabilization of both the A and B fragments was observed (Fig. 2, A and B), and there was a significant reduction in the amount of 37-kDa fragment that accumulated. Although AMP-PNP greatly stabilized the A fragment (which contains the T2 site) and inhibited formation of the 37-kDa peptide by cleavage at T3 in the B fragment, it had little effect on the accessibility of the T4 site to trypsin. AMP-PNP did not qualitatively modify the pattern of tryptic peptides formed from the E2 form of the Ca-ATPase, but both the A and B bands became somewhat stronger as the AMP-PNP concentration was raised to 5 mM (Fig. 2B). This may suggest some additional stabilization through the nucleotide analogue binding to a low affinity site.



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  		FIG. 2.
Effect of AMP-PNP on tryptic cleavage pattern of the E1 and E2 forms of scallop Ca-ATPase. AMP-PNP (pH 7.0) was added to and mixed with DOC-extracted scallop FSR, and digestion with trypsin was carried out as described under "Experimental Procedures." Samples (36 µg) were run in the Laemmli system with a 12.5% separating gel. A, lane 1, markers; lane 2, E1 digest; lane 3, E1 digest + 0.1 mM AMP-PNP; lane 4, E1 digest + 0.5 mM AMP-PNP; lane 5, E1 digest + 1mM AMP-PNP; lane 6, E1 digest + 5 mM AMP-PNP; lane 7, E1 digest + 10 mM AMP-PNP. B, lane 1, E2 digest; lane 2, E2 digest + 0.1 mM AMP-PNP; lane 3, E2 digest + 0.5 mM AMP-PNP; lane 4, E2 digest + 1mM AMP-PNP; lane 5, E2 digest + 5 mM AMP-PNP; lane 6, E2 digest + 10 mM AMP-PNP.

 
Addition of AMP-PCP or ADP to tryptic digests of scallop Ca-ATPase gave very similar effects to AMP-PNP over the same (0.1–1 mM) concentration range. In summary, the overall effect of the nucleotide ligands was to make the pattern of proteolytic products formed from the E1 scallop Ca-ATPase closer to that normally seen in the E2 state, with strong A and B bands on SDS gels and a weak 37-kDa band.

Effect of Orthophosphate on the Tryptic Cleavage of E1—The effect of a range of concentrations of orthophosphate on the tryptic cleavage pattern produced in the presence of Ca2+ (enzyme in the E1 state) is shown in Fig. 3. As the concentration of Pi was increased to 20 mM, there were significant reductions in the amounts of the 37- and 88-kDa fragments formed, i.e. the cleavages at the T3 site in the N domain and the T2 site in the A domain were inhibited. Pi strongly stabilized the A fragment (Met1–Lys504), but as with the nucleotide ligands, there was no significant protective effect on the T4 site. Thus, the Pi-bound E1 form resembled the E2 form in terms of the stability of the A and B fragments to further proteolysis, despite the presence of saturating concentrations of Ca2+. The presence of tripolyphosphate (5 mM) in E1 digests produced similar effects to those seen with Pi.



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  		FIG. 3.
Effect of Pi on the tryptic cleavage pattern of the E1 form of scallop Ca-ATPase. Sodium orthophosphate (pH 7) was added to DOC-extracted scallop SR suspended in the standard E1 digestion medium, and digestion with trypsin was carried out as described in the legend to Fig. 1 and under "Experimental Procedures." Samples (36 µg) were run on a 12.5% Laemmli gel. Lane 1, E1 digest; lane 2, E1 digest + 5 mM Pi; lane 3, E1 digest + 10 mM Pi; lane 4, E1 digest + 20 mM Pi; lane 5, E1 digest + 40 mM Pi.

 
Effect of Orthophosphate on the Tryptic Cleavage of E2— Thus, Pi profoundly affected the tryptic cleavage of the E1 form of the scallop Ca-ATPase. The effect of Pi on the tryptic cleavage of E2 was then examined. As described above, cleavage at the T2, T3, and T4 sites was inhibited when the scallop Ca-ATPase was in its E2 form under the usual conditions in the presence of 0.13 or 0.15 M K+ at pH 7. The presence of 20 mM Pi did not in any way modify the products of the E2 digest made under these standard conditions, as expected. When the E2 form of the Ca-ATPase was digested under identical conditions but with no K+ present, it was rapidly degraded into small fragments (not shown), as anticipated from previous studies that showed that in the absence of K+ the enzyme adopts a loose and open conformation (21). Orthophosphate (20 mM) had no effect on the very extensive proteolysis of E2 in the standard digestion medium lacking K+ at pH 7.

The E2-P Form of Scallop Muscle SERCA Is Very Stable, with a Tightly Folded Conformation—The studies described above on the effect of Pi on tryptic cleavage of E2 had been carried out under conditions that did not promote formation of E2-P. The effect of covalent phosphorylation of E2 was then investigated. Previous studies had shown that membranous scallop Ca-ATPase could be phosphorylated with Pi to yield a form of the enzyme corresponding to the well-studied E2-P form of rabbit SERCA1a (15). Enhancement of steady-state fluorescence (super fluorescence) of TNP-ADP at 532 nm associated with formation of E2-P (28, 29) was used to characterize the affinity of the binding site for Pi on scallop Ca-ATPase involved in formation of E2-P (see "Experimental Procedures"). The intensity increase was half maximal at 3.4 mM added Pi, consistent with the expected affinity of the enzyme for Pi (30).

When the E2 form of the scallop Ca-ATPase was suspended in 20% v/v Me2SO and 15 mM Mg2+ in the absence of Pi, some limited stabilization of the A and B tryptic fragments was observed (Fig. 4, lane 2). However, when 20 mM Pi was present together with 20% v/v Me2SO and 15 mM Mg2+ in the K+-free medium at room temperature to induce formation of the E2-P state, both the A and B fragments became extremely resistant to secondary cleavage by trypsin (Fig. 4, compare lanes 2 and 3). Even after exposure to trypsin for 1/2 h at room temperature, the A and B peptides formed from the E2-P enzyme remained essentially intact. Cleavage was thus effectively restricted to the T1 site with very little secondary proteolysis, whereas after 1/2 h a significant amount of small proteolytic debris had accumulated in the comparable digest of the E2 form. Therefore, although the T1 site remained accessible to trypsin after phosphorylation of the E2 to the E2-P form of scallop SERCA, the rest of the cytoplasmic part of the molecule adopted a conformation that was exceptionally resistant to further attack by trypsin. Because K+ activates hydrolysis of E2-P (31), it was not possible to directly compare the stabilities of E2 and E2-P in the presence of K+. It was noted that the SDS complex of the residual undigested (intact) E2-P form of the Ca-ATPase polypeptide migrated more slowly than the SDS complex of the undigested E2 form (Fig. 4), suggesting that the E2-P-SDS complex was more extended than the E2-SDS complex. It is known from studies of rabbit SERCA1a that there is binding site for ADP on E2-P (29). When 4 mM ADP was included in tryptic digests of the E2-P form of scallop Ca-ATPase (Fig. 4, lane 4), the A fragment became more susceptible to tryptic cleavage and traces of the 22–24 kDa doublet appeared in the digest, indicating that the T4 site had become more exposed. Thus, binding of ADP perturbed the structure of E2-P.



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  		FIG. 4.
Effect of phosphorylation of the scallop Ca-ATPase to its E2-P state on its tryptic digestion pattern. Samples of DOC-extracted scallop FSR were digested with trypsin as described under "Experimental Procedures" at room temperature for 30 min in a medium promoting phosphorylation of the enzyme with Pi. A second sample was digested in a medium otherwise identical but lacking Pi. Samples were electrophoresed in the Tris-glycine system on a 12.5% gel. Lane 1, markers; lane 2, 36 µg of the unphosphorylated E2 form of scallop Ca-ATPase after digestion with trypsin in a medium containing 20% v/v Me2SO, 15 mM MgCl2, 10 mM EGTA-Tris, 50 mM MOPS-Tris, pH 7.0 but no Pi; lane 3, 36 µg of the E2-P phosphorylated form of scallop muscle Ca-ATPase after digestion with trypsin in 20% v/v Me2SO, 15 mM MgCl2, 10 mM EGTA-Tris, 50 mM MOPS-Tris, pH 7.0, plus 20 mM Pi-Tris; lane 4, 36 µg of the E2-P phosphorylated form of scallop muscle Ca-ATPase after digestion with trypsin in 20% v/v Me2SO, 4 mM ADP, 20 mM Pi-Tris, 15 mM MgCl2, 10 mM EGTA-Tris, 50 mM MOPS-Tris, pH 7.0.

 
In the course of these studies, it was found that provided the scallop Ca-ATPase was maintained in the K+-free E2-P phosphorylation medium, it could be kept at room temperature for extended periods of time (>10 days) without loss of activity. Again, as judged by TNP-ADP superfluorescence, both membranous and C12E8-solubilized scallop Ca-ATPase phosphorylated with Pi were very stable with little decay of the signal, provided the samples were kept in the dark between measurements to prevent photobleaching. The stability of scallop FSR in the E2-P state, formed in the absence of Ca2+, K+, and Na+, was in complete contrast to the extremely rapid loss of activity that occurs with the membranous unphosphorylated E2 form of the scallop enzyme under comparable conditions (11, 13). Because inactivation of the unphosphorylated Ca2+-free (E2) scallop Ca-ATPase involves an irreversible loss of the Ca2+-binding sites, phosphorylation of the enzyme in the absence of Ca2+ with Pi may thus stabilize otherwise labile empty Ca2+-binding sites.

In the context of the above results, it was of interest to compare the effect of tryptic digestion of the Ca-ATPase phosphorylated from ATP (+ Ca2+) to that described above where the enzyme had been phosphorylated from Pi (+ EGTA). Because the enzyme was found to be inactivated by secondary tryptic cleavages within the A and B fragments, phosphorylation was carried out before proteolysis in the absence of Mg2+ in the presence of K+, i.e. under conditions where most (>90%) of the phosphorylated enzyme was in the ADP-sensitive form (E1~P) as previously described (15). The pattern of tryptic fragments produced from the E1~P preparation of scallop Ca-ATPase as visualized with Coomassie Blue was indistinguishable from that of the unphosphorylated E1 form of the Ca-ATPase, with significant breakdown of the A and B fragments.

Dependence of Enzyme Activity on ATPMg2 Concentration— Information about the number and type of nucleotide-binding sites on the scallop SERCA was important for interpretation of some of the above results. Many studies of rabbit SERCA1a have suggested the coexistence of catalytic- and regulatory nucleotide-binding sites on that enzyme (33, 34), whereas there is evidence for only one type of site on the SERCA of the cold-resistant wood frog (35). Thus, the dependence of the Ca2+-activated ATPase activity of deoxycholate-extracted scallop muscle SERCA vesicles on ATP concentration was examined according to Eadie-Hofstee (36), where the activity (vo) is plotted against the ratio of activity to ATPMg concentration as shown in Equation 1.

(Eq. 1)

The graph displayed two limbs, one with a slope corresponding to an apparent Michaelis constant KH of 0.29 mM, which extrapolated to give a kcat of 368.5 min–1, and the other to a Km of 4.6 µM and a kcat of 92.4 min–1, (assuming a molecular mass of 110 kDa and that the Ca-ATPase constituted 95% of the total protein in the deoxycholate-extracted vesicles used in the experiments.) The central portion of a Hill plot of the data had a slope of nH = 0.6, while the double reciprocal (Lineweaver-Burke) plot was convex upwards (not shown). These results strongly resembled those obtained with rabbit SERCA1a (37, 38), which have been interpreted in terms of a high affinity catalytic nucleotide-binding site and a low affinity regulatory site.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The affinities of the catalytic- and regulatory nucleotide-binding sites on SERCa1a for ATP (2–5 µM and 0.3–1 mM, respectively, (38) are very similar to the Km and KH of scallop Ca-ATPase found here. There is much evidence for two separate binding sites for nucleotide on each Ca-ATPase polypeptide chain (8, 39, 40), with one of the sites functioning as an allosteric regulator site and the other as the active (catalytic) site. Thus, there are two potential nucleotide-binding sites on the scallop Ca-ATPase that have to be taken into consideration with regard to stabilization of the scallop A and B fragments in the E1 form of the Ca-ATPase by Pi, AMP-PNP, AMP-PCP, and ADP.

The affinity of the catalytic site for AMP-PNP on rabbit SERCA1a (Kd ~ 75–90 µM (36, 41, 42) is in keeping with the concentration of that ligand that stabilized the A and B fragments in tryptic digests of the E1 form of scallop Ca-ATPase. Electron paramagnetic resonance experiments using spin-labeled rabbit SERCA1a (43) suggest that AMP-PCP binds to two sites on rabbit SERCA1a with dissociation constants of 50 and 650 µM. The lower value fits with stabilization of the A and B fragments of the scallop Ca-ATPase through AMP-PCP binding to the catalytic site. Although metal-free ADP binds only weakly to the phosphorylated E1~P(Ca2+)2 form of rabbit SERCA1a (Kd values ~0.73 mM) (44), it binds more strongly to the unphosphorylated enzyme with reported Kd values of 12–50 µM (45). These are consistent with stabilization of the A and B fragments in digests of the E1 form of scallop Ca-ATPase by ADP being bound to the catalytic site.

In the case of the rabbit enzyme, E1 and E2 have the same affinity for Pi (5–10 mM) (30), whereas the Kd of the catalytic site for Pi on the E2 form of scallop Ca-ATPase was found in the studies reported here to be 3.4 mM on the basis of TNP-ADP fluorescence measurements. These values are in the concentration range where stabilization by Pi of the A and B fragments of the E1 form of the scallop enzyme was manifested, so that binding of Pi to E1 is likely to be mediated through its occupation of some part of the catalytic site. Occupation of only that part of the active site that interacts with the very small orthophosphate ligand was sufficient to cause a very substantial reorganization of the three subdomains (A, N, and P) that comprise most of the cytoplasmic region of the scallop Ca-ATPase. Although the nucleotides were effective at significantly lower concentrations than Pi, this probably primarily reflects their higher binding affinities. Thus, binding of the adenosine moiety was not essential for stabilization of the E1 form of scallop Ca-ATPase. Binding of Pi or the polyphosphate moiety of nucleotide to the Ca-ATPase stabilizes a conformation where the cytoplasmic domains of the enzyme lie in close proximity to one another so that tryptic cleavages are inhibited in the A and N domains.

There is already good evidence that binding of nucleotide can modify the conformation of rabbit skeletal muscle and scallop adductor muscle SERCA. In particular, the rate of a conformational change associated with the binding of Ca2+ is increased by ATP, and binding of ATPMg changes the conformation of the enzyme to one that is activated for phosphorylation (46). In fact, ADP, AMP-PCP, and AMP-PNP accelerate Ca2+ binding in a pH-dependent manner (47), and changes in the amide I and II regions of the IR spectrum show that binding of nucleotide modifies the conformation of rabbit SERCA1a (48). In the case of the scallop Ca-ATPase, addition of 5 mM ATPMg2– to the E2 form of the scallop Ca-ATPase leads to ~4 additional thiol groups becoming less reactive toward the thiol reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB), whereas addition of 6 mM ATP to the E1 form in the absence of Mg2+ causes ~5 additional thiol groups to become inaccessible to DTNB (21).

All of the ligands that stabilized the A and B fragments in Ca2+-saturated (E1) scallop Ca-ATPase against further digestion by trypsin induced a form of the enzyme with some conformational features in common with the Ca2+-free E2 state, despite Ca2+ being bound. However, there is no evidence that, for example, AMP-PNP lowers the affinity of the enzyme for Ca2+, thereby producing an E2-like state. On the contrary, occupation of the nucleotide-binding site has been reported to increase, not decrease, affinity of the Ca-ATPase for Ca2+ (48). The T4 site on E1 was not protected by any of the agents, so that the modified form of E1 induced by binding of substrate analogues or Pi was not identical to E2 and possessed a unique structure. Hence, although binding of Ca2+ can profoundly modify interactions among the cytoplasmic subdomains, those regions of the enzyme still retain some independence from the transmembrane portion of the enzyme in their response to the binding of ligands.

It has been pointed out that the binding of Ca2+ alone is not sufficient to position the nucleotide substrate close enough to Asp351 for transfer of the {gamma}-phosphoryl group (49). The results reported here suggest that the additional reorganization of the catalytic center necessary for this to happen may originate in conformational changes caused by occupation of the active site, an example of the induced fit phenomenon. The occlusion of potentially sensitive sites toward trypsin that occurs when substrate analogues are bound to the catalytic nucleotide-binding site in the E1 form of the Ca-ATPase may be related to the closing of the residual distance between Asp350 (scallop sequence) and the {gamma}-phosphoryl group of ATP.

Recently, it was found that treatment of rabbit SERCA1a with the phosphate transition state analogues F– and orthovanadate caused the enzyme to become extremely resistant to the action of several proteases (50, 51). In the current study, formation of the E2-P covalent adduct by addition of Pi to the E2 form of the scallop SR membranes suspended in a K+-free medium prevented an otherwise rapid degradation of the A and B fragments by trypsin. This behavior suggests an unusually condensed conformation for the scallop enzyme in its E2-P state; however, because the E2-P-SDS complex had a lower mobility than the E2-SDS complex, overall the E2-P form may not be folded into a more globular shape than E2. The unphosphorylated E2 form of scallop Ca-ATPase loses activity very rapidly and is highly susceptible to proteolysis in the absence of K+, yet formation of the aspartyl phosphate at the active site was able to lock the enzyme into a conformation that was both structurally and functionally exceedingly stable. Thus, the effect of the covalent modification was propagated throughout the cytoplasmic domains of the enzyme.

The resistance of the E2-P form of scallop Ca-ATPase toward trypsin and the great stability of its enzyme activity compared with E2, both under conditions where K+ are absent, are very likely to have a common origin in the unusual state of the active site in E2-P. The synergistic binding of Pi and Mg2+ to E2 is endothermic, but a substantial entropy increase associated with the release of a large number of water molecules from the active site and the hydration shell of Pi allows the process to be driven thermodynamically (27, 52). The activity of water at the catalytic site in the E2-Mg2+-Pi ternary complex is therefore very low; this very non-polar environment promotes spontaneous formation of the acyl phosphate from Pi and the {beta}-carboxyl side chain of Asp351 and then protects it from hydrolysis (3). The very hydrophobic nature of the active site in E2-P may underlie its great stability as well as the reported differences in quaternary and tertiary structure between E2-P and E2 (15, 53–55).

Stabilization by Pi of E1 in a form where Ca2+-sensitive tryptic cleavage sites on the A and N domains are occluded toward trypsin and the great resistance of E2-P toward trypsin are consistent with what is known about the binding site for Pi. All three cytoplasmic domains may have to be intimately juxtaposed to form the site in combination with the hinge region. Thus, atomic models suggest that formation of the non-polar cavity in E2-P may involve movement of the A domain close to the catalytic site (39). Support for such a structure comes from studies using proteinase K digestion of SERCA1a (56) and chymotryptic digestion of the Na, K-ATPase (57). Further strong evidence that the A domain contributes to the Pi-binding site derives from site-directed mutagenesis and chemical modification studies of Gly233 and Arg198, both located in L23, (58, 59). The close proximity of Glu485-Asp489 to Thr170-Leu172 in the E2 crystal structure of SERCA1a suggests that the N domain may also be involved in the binding of Pi, and there is strong evidence that the C-terminal part of the P domain of SERCA1a is needed for Pi to bind (60). The h1-h2 hinge connecting the N and P domains must participate in the active site in E2-P because it contains Asp351. There is also good evidence that the hinge is directly involved in the binding of Pi, because the phosphate transition-state analogue orthovanadate binds close to Thr353 in h1 (61), and the 601DPPR motif in h2 provides ligands for the binding of Pi (62). Formation of the Pi-binding site thus appears to involve all the major structural elements of the cytoplasmic region of the Ca-ATPase. Orthophosphate or the phosphate moieties of nucleotides may therefore act to draw the cytoplasmic domains and the hinge together and so stabilize a compact structure resistant to trypsin.

In conclusion, large-scale structural changes can be initiated in the Ca-ATPase not only by the binding and release of Ca2+ but also by interaction of phosphate groups with the active site. Another example of a major structural reorganization in a protein associated with Pi binding/release is the very large conformational change that occurs when Pi dissociates from the S-1 head of myosin after hydrolysis of ATP (32).


   FOOTNOTES
 
* This work was supported by a grant from the Central Research Committee of the School of Medicine, Southern Illinois University (to P. M. D. H.). 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. Back

{ddagger} Supported by R01 GM56960. Present address: Skirball Institute, NYU Medical Center, 540 First Ave., New York, NY 10016. Back

§ Present address: Open System Div., Information Builders Inc., Two Penn Plaza, 28th Floor, New York, NY 10121. Back

To whom correspondence should be addressed. Tel.: 618-453-6469; Fax: 618-453-6440; E-mail: phardwicke{at}siumed.edu.

1 The abbreviations used are: SERCA, sarco(endo)plasmic reticulum ATPase; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; AMP-PCP, adenosine 5'-({beta}, {gamma}-methylene) triphosphate; AMP-PNP, 5'-adenylylimidodiphosphate; DOCNa, sodium deoxycholate; FSR, fragmented sarcoplasmic reticulum; MES, 4-morpholineethanesulfonic acid; MOPS, 3-(N-morphilino)propanesulfonic acid; Pi, orthophosphate; SR, sarcoplasmic reticulum; A, N, and P, actuator, nucleotide, and phosphorylation domains, respectively; TNP-ADP, 2',3'-O-(2,4,6-trinitrocyclo-hexyldienylindine) adenosine 5'-diphosphate; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone. Back

2 Because the scallop Ca-ATPase lacks a residue corresponding to Thr22 of rabbit SERCA1a, b (26), over almost all of the sequence (from Gly22 until an extra residue is inserted at position 987 (Cys987)) residue position numbers for the scallop ATPase are one less than the corresponding residue number in SERCA1a, b. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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