Crosslinking the active site of sarcoplasmic reticulum Ca2+-ATPase ...

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6437-6441, August 1991

Biochemistry

Crosslinking the active site of sarcoplasmic reticulum Ca2+-ATPase completely blocks Ca2+ release to the vesicle lumen (Ca2+ transport/uncoupling/conformational change/energy transduction/muscle)

DAVID B. MCINTOSH*, DAVID C. Ross*, PHILIPPE CHAMPEILt,

AND

FLORENT GUILLAINt

*Medical Research Council Biomembrane Research Unit and Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa; and tService de Biophysique, Departement de Biologie, Centre d'Etudes Nucleaires Saclay, 91191 Gif-sur-Yvette Cedex, France

Communicated by Paul D. Boyer, April 15, 1991

Intramolecular crosslinking of the active site ABSTRACT of the sarcoplasmic reticulum Ca2+-ATPase with glutaraldehyde results in substantial inhibition of ATPase activity and stabilization of the ADP-sensitive El-P(2Ca) intermediate (E, enzyme) with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. We show here, using conditions of low passive vesicle permeability and absence of ADP, that Ca2+ "deoccludes" more rapidly than it leaks out of the vesicle lumen. Deocclusion is paralleled by dephosphorylation. Therefore, turnover of crosslinked El -P(2Ca) (-5 nmol/min per mg of protein at 25QC) involves Ca2+ release to the vesicle exterior and concomitant phosphoenzyme hydrolysis. Ca2' release to the lumen, the normal pathway, is apparently blocked completely. In the presence of ADP, Ca21 is also released to the vesicle exterior, and this release is coupled to the synthesis of ATP. The results suggest that a tertiary structural change at the active site follows phosphorylation and is an absolute requirement for Ca2+ release from the native enzyme to the vesicle lumen.

Ca2" transport of sarcoplasmic reticulum (SR) is carried out by a membrane-embedded Ca2"-ATPase. Investigation of the partial reactions of the pump cycle has shown that translocation of Ca2" takes place in the first part of the cycle and can be divided into three main steps, involving binding on the cis side of the membrane, occlusion within the protein, and release to the trans side or vesicle lumen (1-7). These events at the transport site are coupled to chemical changes at the active site such as the change in the reactivity of an aspartic residue to ATP, phosphorylation of the residue to an ADPsensitive form, and then a change to an ADP-insensitive phospho form (8-10). The importance of active-site movements and the nature of the communication between the transport site and the active site have been explored by determining the functional consequences of introducing an intramolecular crosslink at the active site (11-13). Glutaraldehyde reacts with the active site of the Ca2+-ATPase to initially produce a crosslink between tryptic fragments Al and B (11, 12). Formation of the crosslink is inhibited by nucleotide binding or by phosphorylation to the ADPinsensitive E2-P catalytic intermediate (E, enzyme). The crosslink inhibits formation of E2-P in both directions of catalysis and stabilizes the ADP-sensitive E1-P(2Ca) intermediate with occluded Ca2+ ions (13). These results suggest that Ca2' release to the vesicle lumen and a change from an ADP-sensitive to an ADP-insensitive phospho form are accompanied by a tertiary structural shift at the active site, which is inhibited by the crosslink. In this study, we have investigated whether the crosslinked ATPase pumps

Ca2+ or not. We find that the crosslink

completely blocks the normal pathway of Ca2+ release to the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

lumen and that the enzyme is forced to follow an uncoupled pathway of catalysis with Ca2l deoccluding to the vesicle exterior on hydrolysis of the phosphoryl group. This suggests that a change in the tertiary structure in the vicinity of the nucleotide binding site following phosphorylation is an absolute requirement for Ca2" release to the lumen. The findings support a mechanism of energy transduction in which coupling of active-site events and directional cation movement includes a type of mechanical gating.

MATERIALS AND METHODS Glutaraldehyde (grade 1, 25% solution), ATP, acetyl phosphate, and Ca2+ ionophore A23187 were purchased from Sigma. Acetyl [32P]phosphate was synthesized as described by Bodley and Jencks (14). SR vesicles were prepared from rabbit skeletal muscle (15). In some cases, 0.1 mg of amylase (Sigma) was added to the preparation during the initial homogenization step (concentration, 1 ,g/ml). This procedure eliminated the presence of phosphorylase and a protein of ==140 kDa in the final SR preparation. Crosslinking of the ATPase (0.5 mg of SR protein per ml) was carried out at 25°C in 50 mM Mops/triethanolamine, pH 8.1/1 mM MgCl2/10 mM KCI/0.2 M sucrose/150 ,uM glutaraldehyde (37,000-fold dilution of 25% solution) (13). A reaction time of 1 hr was taken as optimal for formation of the crosslinked ATPase. After this time, the preparation consisted of -85% ATPase specifically crosslinked at the active site, 12% intermolecularly crosslinked ATPase (oligomers), and 3% uncrosslinked ATPase. Ca2+ uptake and phosphoenzyme determination were performed at 20°C using [45Ca]CaCl2 and acetyl [32P]phosphate, respectively, combined with filtration on Millipore 0.45-Am filters (HAWP 025 00). The assays were initiated by the addition of either ATP, acetyl phosphate, or acetyl [32P]phosphate and terminated by filtration and immediate washing with two 3-ml aliquots of ice-cold wash medium (50 mM Mes/Tris, pH 6.0/100 mM KCl/5 mM MgCl2/0.5 mM EGTA). In some cases, uptake was terminated at predetermined times by addition of EGTA with or without ADP, or

with unlabeled Ca2+, and the release period was followed by filtration and washing. The time was taken as that from the addition of substrate to the initiation of the washing procedure. The filters were assayed for 45Ca and 32P radioactivity in Instagel (Packard). It was assumed that all the Ca2+dependent 32P radioactivity remaining after the wash represented ATPase phosphoprotein intermediate (16). Measurement of ATP synthesis and Pi release were carried out as follows. Glutaraldehyde-treated SR vesicles were incubated with acetyl [32P]phosphate in a medium and for a time indicated in the legend to the appropriate figure. The suspension was then filtered through a Millipore 0.45-,um filter unit (Millex-HA) with positive pressure from a syringe. Abbreviation: SR, sarcoplasmic reticulum.

6437

6438

Biochemistry: McIntosh et al.

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Proc. Natl. Acad Sci. USA 88 (1991)

By changing of syringes as rapidly as was manually possible, the filter was quickly washed with 6 ml of ice-cold wash medium (see above) and then perfused with 5 ml of 100 mM Mes/Tris, pH 6.0/100 mM KCI/5 mM MgCl2/1 mM ADP/5 mM EGTA at 20°C for various times. The filtrates were mixed directly with 5 ml of ice-cold 0.2 mM ATP/0.2 mM Pi. The mixture was then placed on a small column (0.5 x 1.5 cm) of Dowex AG1-X4 (Bio-Rad). Pi was eluted with 10 ml of 50 mM MgCl2, and ATP with 10 ml of 0.5 M HCl. Measurement of 32P was by Cerenkov radiation. Recovery of ATP was determined by absorbance at 259 nm.

RESULTS We tried two strategies for determining whether the crosslinked ATPase pumps Ca> to the vesicle lumen or not. One was to extensively modify the ATPases, in an attempt to eliminate the contribution of the uncrosslinked ATPases, and then determine whether Ca2+ was pumped or not. The other was to use less extensively modified preparations and determine whether the occluded Ca2+ within the crosslinked E1-P(2Ca) catalytic intermediate was released to the exterior or the interior of the vesicle during enzyme turnover. The results of the first approach are shown in Fig. 1. Unmodified preparations exhibited rapid Ca> uptake that was completely eliminated by the presence of A23187, a Ca2+ ionophore (Fig. 1, circles). Reaction with glutaraldehyde for 1 hr resulted in partial inhibition of Ca2+ uptake measured in the absence of ionophore and the association of -7 nmol of

Ca2+ per mg of vesicle protein in its presence (Fig. 1, triangles). The latter Call represents that occluded within E1-P(2Ca) (13). After 4 hr of reaction, the rate of Ca>2 uptake (v value minus V value) approximately equaled the rate of turnover of the crosslinked enzyme (K). After 6 hr (data not shown), the rate of transport was further diminished. The question whether the transport was due to crosslinked ATPases or residual uncrosslinked ATPases is impossible to answer. The large difference in turnover rates of the crosslinked and uncrosslinked ATPases means that even very small amounts of the latter are significant. The long reaction times contribute uncertainties regarding the Ca2> leak rate and the effect of intermolecular crosslinking. The other approach, involving less complete reaction with glutaraldehyde and determination of the direction of Ca2> deocclusion, was more successful as it did not depend on eliminating the contribution of the uncrosslinked ATPases. In essence what was required was to find conditions under which Ca>+ release from the crosslinked enzyme was significantly faster than that from the vesicle lumen. Such conditions were found at low pH and at high Mg> concentrations. First, the effect of crosslinking on the passive leak of Ca2> was examined (Fig. 2). 45Ca2> efflux from control vesicles into medium with EGTA (0) or 4Ca2> (free concentration, -0.1 mM Ca+; o) was biphasic, with most of the Ca2> Control

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FIG. 1. Effect of crosslinking on Ca2+ transport and Ca2+ occlusion. The time course of Ca2+ retention was measured at 20°C with native vesicles (circles) or after 1 hr (triangles), 2 hr (squares), and 4 hr (inverted triangles) of preincubation of SR vesicles in crosslink medium with glutaraldehyde. The assay medium contained 100 mM Mops/Tris (pH 7.0), 100 mM KCl, 5 mM MgCl2, 0.1 mM 45CaC12, 0.2 mM ATP, and 0.05 mg of SR protein per ml, in the absence (solid symbols) or in the presence (open symbols) of 4% (wt/wt of protein) A23187. The turnover rate of the crosslinked ATPase was determined following addition of 10 mM EGTA after 2 min of ATP-dependent sequestration in the presence of A23187 by SR vesicles treated for 4 hr with glutaraldehyde (open diamonds).

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Time, min FIG. 2. Effect of crosslinking on passive Ca2+ leak. SR vesicles were preincubated for 1 hr without (Upper) or with (Lower) glutaraldehyde in the presence of 1 mM 45CaC12 instead of the usual 1 mM MgCI2. The time course of Ca2+ efflux was followed at 20°C by diluting the suspension 10-fold into 100 mM Mes/Tris, pH 6.0/100 mM KCI/5 mM MgCI2 with either 5 mM EGTA (e) or 5 mM '4CaCl2 plus 6 mM EGTA (free Ca2, -0.1 mM; o) and taking timed aliquots for filtration. The earliest time point was 10 s.


Proc. Natl. Acad. Sci. USA 88 (1991)

released during the slow phase. Medium Ca2" significantly slowed the release of this 45Ca2", as has been found by others (17, 18). Glutaraldehyde treatment had no effect on the kinetics of release (Fig. 2 Lower). The relationship between the load, the biphasic nature of the Ca2" efflux, and the dependence on external Ca2+ concentration was examined in more detail following active loading of control vesicles with acetyl phosphate (Fig. 3), conditions under which the direction of Ca2' release from the crosslinked enzyme would be determined. Efflux into -EGTA medium resulted in biphasic kinetics at each of the three Ca2+ loads investigated (Fig. 3A). The amplitude of the initial, more rapid, phase increased with load but the time dependence of both phases was unaffected and was similar to that found above, after passive loading. In contrast, efflux of radioactive tracer following addition of nonradioactive Ca2+ was monophasic, or almost so, and the rate constants ofrelease increased with load (Fig. 3B). While the reasons for the different kinetics of efflux under non-turnover and turnover conditions may be complex, the important points for this study are that (i) the majority of the luminal Ca2+ is released into EGTA medium with a -

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Time, min FIG. 3. Ca2l release from native vesicles following active loading with acetyl phosphate. SR vesicles (0.05 mg of protein per ml) were incubated at 200C in 50 mM Mes/Tris, pH 6.0/100 mM KCI/5 mM MgCI2/0.1 mM [45Ca]CaCl2/0.05 mM acetyl phosphate for 0.5 min (A), 2 min (o), and 4 min (e). Then either 10 mM EGTA (A) or 5 mM plus 6 mM EGTA (B) was added and the suspensions were filtered at timed intervals. The earliest time point was 10 s.

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6439

relatively slow rate constant (-0.06 min') and (ii) virtually all of the accumulated radioactive tracer leaks out even more slowly (especially at low loads) into a medium containing micromolar free Ca2' during sustained enzyme turnover. The experiments concerned with the direction of Ca2+ release from the crosslinked ATPase are shown in Fig. 4. The effect of the addition of EGTA is examined first (Fig. 4A). When acetyl phosphate was added to the crosslinked ATPase in the presence of A23187, there was a time-dependent increase in occluded Ca2+ (o) and phosphoenzyme (n) to a maximum of approximately 5 and 2.5 nmol/mg of protein, respectively. This represents formation of the crosslinked E1-P(2Ca) intermediate. Addition of EGTA resulted in Ca2+ release (A) and dephosphorylation (K) with the same rate constants (0.63 min'). In the absence of the Ca2+ ionophore, the initial kinetics of Ca2+ sequestration (0) and phosphorylation (A) were similar to those in its presence, but after 1 min there was a linear uptake of Ca2+ into the vesicle lumen, without a change in the phosphoenzyme level. Addition of EGTA after 1 min resulted in monophasic Ca2' release (A) with the same rate constant as that which occurs in the presence of ionophore. Of particular significance was the lack of any slow phase that could be attributed to leakage of luminal Ca2 . Clearly, the occluded Ca2+ was released directly to the vesicle exterior. Addition of EGTA after 15 min of uptake resulted in the biphasic release of Ca2+ (A), with the faster phase occurring with a rate constant of 0.76 min-1, similar to that of Ca2+ deocclusion in the presence of ionophore, and the slower phase occurring at 0.047 min-, similar to the rate constant of passive efflux of the majority of the luminal pool of Ca2+, determined above. The slightly greater amount of Ca2+ over and above the occluded Ca2+ (1-2 nmol/mg of protein) in the faster phase was accounted for by the more rapidly released pool of Ca2+ seen in control vesicles. The rate of dephosphorylation following the addition of EGTA after 15 min of uptake (*) paralleled the faster phase of Ca2+ release and dephosphorylation in the presence of ionophore. Again the direction of deocclusion was to the vesicle exterior. The direction of Ca2+ deocclusion was even more obvious when CaEGTA was added to the medium (50-fold dilution of 45Ca2+ with 40Ca2+, with the free Ca2+ concentration unchanged), conditions under which passive efflux is slower and the enzyme continues to turn over (Fig. 4B). Ca2+ release in the absence of A23187 (A) after 1 min of sequestration (k = 0.49 min') was the same as that in its presence and the same as occurred in EGTA medium (compare with Fig. 4A). Clearly the decrease in passive permeability did not affect the release of Ca2+ from the crosslinked ATPase. After 15 min of uptake, the biphasic release of Ca2+ was obvious and could only be attributed to the fast release of Ca2+ from the crosslinked enzyme and the slow passive efflux of luminal Ca2 , pumped in by the uncrosslinked species. Dilution of 45Ca2+ with 4OCa2+ had little effect on phosphoenzyme levels (O, *), as expected. Assignment of the fast phase of Ca2+ release from glutaraldehyde-treated vesicles to exterior deocclusion from the phosphorylated ATPase was confirmed by its sensitivity to ADP. Although the crosslink greatly slows the rate of nucleotide-dependent phosphoryl transfer reactions, the crosslinked E1-P(2Ca) intermediate still retains its complete ADP-sensitivity (13). Fig. 4C shows that addition of EGTA plus ADP to the vesicles, measured in the absence of A23187, resulted in the acceleration of dephosphorylation and deocclusion coupled with nearly stoichiometric synthesis of ATP, as expected for reversal of the cycle. The direction of Ca2+ release was to the vesicle exterior and again the slow phase was ascribable to Ca2' release from the lumen. That the direction of Ca2+ release was correctly deduced in this case, where the direction of release was known to be to the


6440

Proc. Natl. Acad. Sci. USA 88 (1991)

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C FIG. 4. Kinetics of Ca2+ release and phosphoenzyme levels of crosslinked preparations. SR vesicles were treated with glutaraldehyde for 1 hr under standard conditions. Ca2" retention (circles, triangles) and phosphoenzyme (EP) levels (squares, diamonds) were assayed at 20TC in 100 mM Mes/Tris, pH 6.0/100 mM KCI/5 mM MgCl2/0.1 mM 45CaCl2/0.03 mM acetyl [32P]phosphate in the absence (solid symbols) or presence (open symbols) of 4% (wt/wt of protein) A23187. At the indicated times, 5 mM EGTA (A), 5 mM 4CaC12 plus 6 mM EGTA (B), or 1 mM ADP plus 5 mM EGTA (C) was added and aliquots were removed at intervals for filtration. In C, the amount of ATP synthesized (x) and Pi released (+) are also indicated. (Insets) Semilogarithmic plots ofthe time course of Ca2+ efflux after 15 min, showing the experimental data and that following subtraction of the slower component. The rate constants for the fast and slow phases (obtained from the drawn straight lines) are 0.76 min- and 0.047 min' (A), 0.40 min' and 0.011 min- (B), and 0.016 min- (C, slow phase only).

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DISCUSSION This study shows unequivocally that the SR Ca2+-ATPase, when crosslinked at the active site with glutaraldehyde, cannot release Ca2+ in the direction of the vesicle lumen, as in the case of the native ATPase, but does release Ca2+ toward the vesicle exterior, even in the absence of ADP. Deocclusion occurs concomitantly with hydrolysis of the phosphoenzyme and therefore represents an uncoupled pathway of substrate hydrolysis. Reaction of the crosslinked El-P(2Ca) intermediate with ADP yields ATP and the Ca2+ deoccludes to the vesicle exterior. The steps catalyzed by the crosslinked ATPase are shown with bold arrows in Scheme I (ref. 13 and the present results). The prohibited steps, which are catalyzed by the native enzyme, are those involving the E2-P intermediates. These intermediates are those with the Ca21 sites oriented toward the vesicle lumen (13). In the forward direction of catalysis, the block forces the enzyme to follow the uncoupled pathway, involving direct hydrolysis of the ADP-sensitive E1-P(2Ca) intermediate (step 7). The results have important implications for the mechanism of energy transduction at the crucial translocation step (step 3). They suggest that the crosslink blocks an essential tertiary movement at the active site that is required for orienting and

releasing the cations at the transport site in the direction of the lumen. Realignment of the ATP-binding domain and the phosphorylation domain by stabilization of specific bridging interactions could shift the position of stalk helices linking the active site and the membrane-located transport site (19-21). 2CaOj

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Biochemistry: McIntosh et al. Another important conclusion is that the vectorial switch, which dictates that the direction of Ca2l deocclusion is to the lumen and not to the vesicle exterior, occurs after phosphorylation and not upon phosphorylation. The demonstration of a stable occluded form of the phosphoenzyme with no access for the Ca2l to the lumen shows that Ca2l release in this direction requires a further event (step 3). The difficulty experienced in detecting the E2-P.2Ca intermediate from kinetic studies of the native enzyme (22) suggests that it is more unstable than some of the other, more prominent intermediates. Its instability can also be deduced from the destabilization of the E2-vanadate complex, considered to be similar to E2-P, by Ca2' binding to luminal low-affinity sites (23, 24). Ca2' release from E2-P.2Ca might also be of a dynamic nature, corresponding to transient opening of a luminal gate (25). The demonstration of a definite occluded species in detergent-solubilized Ca2+-ATPase using CrATP, which also appears to "freeze" the active site (26), supports the view that phosphorylation per se is not sufficient to allow dissociation to the lumen. Hydrolysis of the ADP-sensitive phosphoenzyme violates one of the coupling rules ofJencks (27) and has been expected to be a minor pathway of the native enzyme. Stahl and Jencks (22) estimated the rate constant of the uncoupled pathway to be