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Interaction of Nucleotides with Asp351 and the Conserved Phosphorylation Loop of Sarcoplasmic Reticulum Ca2+-ATPase

1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês

10.1074/jbc.274.36.25227

ISSN

1083-351X

Autores

David B. McIntosh, David G. Woolley, David H. MacLennan, Bente Vilsen, Jens Peter Andersen,

Tópico(s)

GABA and Rice Research

Resumo

The nucleotide binding properties of mutants with alterations to Asp351 and four of the other residues in the conserved phosphorylation loop,351DKTGTLT357, of sarcoplasmic reticulum Ca2+-ATPase were investigated using an assay based on the 2′,3′-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N3-ATP) photolabeling of Lys492 and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca2+, mutations removing the negative charge of Asp351 (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N3-ATP and MgATP with affinities 20–156-fold higher than wild type (KD as low as 0.006 μm), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N3-ATP 2–1000- and 1–6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys492. In the absence of Ca2+, mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg2+ and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca2+(KD = 0.001 μm), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca2+ occlusion due to defective CrATP binding. The nucleotide binding properties of mutants with alterations to Asp351 and four of the other residues in the conserved phosphorylation loop,351DKTGTLT357, of sarcoplasmic reticulum Ca2+-ATPase were investigated using an assay based on the 2′,3′-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N3-ATP) photolabeling of Lys492 and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca2+, mutations removing the negative charge of Asp351 (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N3-ATP and MgATP with affinities 20–156-fold higher than wild type (KD as low as 0.006 μm), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N3-ATP 2–1000- and 1–6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys492. In the absence of Ca2+, mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg2+ and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca2+(KD = 0.001 μm), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca2+ occlusion due to defective CrATP binding. 2′,3′-O-(2,4,6-trinitrophenyl)-8-azido-adenosine triphosphate trinitrophenyl adenylyl β,γ-methylene triphosphate β,γ-bidentate chromium(III) complex of ATP major conformational states of Ca2+-ATPase 3-(N-morpholino)propanesulfonic acid N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid high pressure liquid chromatography 2-{[2-hydroxy-1,1-bis(hy-droxymethyl)ethyl]amino}ethanesulfonic acid tetramethyl ammonium hydroxide The Ca2+-ATPase of sarcoplasmic reticulum is a 10-transmembrane helix Ca2+/H+ pump that hydrolyzes ATP through transient formation of an aspartyl phosphorylated intermediate (1Hasselbach W. Makinose M. Biochem. Z. 1961; 333: 518-528PubMed Google Scholar, 2MacLennan D.H. Brandl C.J. Korzak B. Green N.M. Nature. 1985; 316: 696-700Crossref PubMed Scopus (805) Google Scholar, 3Levy D. Seigneuret M. Bluzat A. Rigaud J-L. J. Biol. Chem. 1990; 265: 19524-19534Abstract Full Text PDF PubMed Google Scholar, 4Degani C. Boyer P.D. J. Biol. Chem. 1973; 248: 8222-8226Abstract Full Text PDF PubMed Google Scholar). The phosphorylated aspartate residue (Asp351) and the binding site for MgATP are located in the large cytoplasmic domain of the pump protein, whereas the Ca2+ transport sites are in the membrane domain (5MacLennan D.H. Rice W.J. Green N.M. J. Biol. Chem. 1997; 272: 28815-28818Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). It is a well documented property of the pump that binding of Ca2+at the transport sites is required to activate phosphoryl transfer from ATP to Asp351. However, the long range intramolecular interaction between the Ca2+ sites and the nucleotide binding site that triggers formation of a transition state for phosphoryl transfer and the nature of this transition state are not well understood. Electrostatic interactions in the vicinity of the phosphoryl groups of ATP and Asp351 and other catalytic residues may be expected to dominate during ATP binding and in the transition state and possibly drive changes in the transport sites (6Pedersen P.A. Rasmussen J.H. Jorgensen P.L. Biochemistry. 1996; 35: 16085-16093Crossref PubMed Scopus (44) Google Scholar). The unphosphorylated Ca2+-ATPase exists in a Ca2+- and pH-dependent equilibrium (7Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar) of several (E1/E2) conformational states (Scheme 1) that appear to interact differently with ATP.E2H3↔E2H↔E1↔E1Ca↔E1Ca2SCHEME1Although in the presence of Mg2+ all of the states indicated in Scheme 1 exhibit rather high affinities for ATP (KD in the range 0.5–20 μm) (8Lacapère J-J. Guillain F. Eur. J. Biochem. 1993; 211: 117-126Crossref PubMed Scopus (25) Google Scholar, 9Lacapère J-J. Bennett N. Dupont Y. Guillain F. J. Biol. Chem. 1990; 265: 348-353Abstract Full Text PDF PubMed Google Scholar), only the E1Ca2 state is primed for transfer of the γ-phosphoryl group to Asp351. As counterions to Ca2+ (3Levy D. Seigneuret M. Bluzat A. Rigaud J-L. J. Biol. Chem. 1990; 265: 19524-19534Abstract Full Text PDF PubMed Google Scholar), protons bind to the transport sites in place of Ca2+, stabilizing the E2 conformation. The fully protonated E2 form (E2H3 in Scheme 1) is phosphorylated by Pi at Asp351 when the pump works in the reverse mode but cannot be phosphorylated by ATP. Since ATP accelerates Pi binding (10Pickart C.M. Jencks W.P. J. Biol. Chem. 1984; 259: 1629-1643Abstract Full Text PDF PubMed Google Scholar) and dephosphorylation (11Champeil P. Riollet S. Orlowski S. Guillain F. Seebregts C.J. McIntosh D.B. J. Biol. Chem. 1988; 263: 12288-12294Abstract Full Text PDF PubMed Google Scholar), modulates Pi ⇌ HOH exchange (12McIntosh D.B. Boyer P.D. Biohemistry. 1983; 22: 2867-2875Crossref PubMed Scopus (78) Google Scholar), and binds fairly tightly to the vanadate complexed E2 form (13Andersen J.P. Moller J.V. Biochim. Biophys. Acta. 1985; 815: 9-15Crossref PubMed Scopus (37) Google Scholar), E2H3 must be able to bind ATP without preventing the access of Pi to Asp351, suggesting that the γ-phosphoryl group of the bound ATP is at some distance from Asp351 in this conformation, in contrast to the E1Ca2 state. A change in the interaction of bound nucleotide with Asp351 related to enzyme activation by Ca2+ is clearly demonstrated with TNP-8N3-ATP1 that has been covalently attached to Lys492 by light activation (14McIntosh D.B. Woolley D.G. Berman M.C. J. Biol. Chem. 1992; 267: 5301-5309Abstract Full Text PDF PubMed Google Scholar, 15McIntosh D.B. Woolley D.G. J. Biol. Chem. 1994; 269: 21587-21595Abstract Full Text PDF PubMed Google Scholar, 16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Tethering the nucleotide still permits Ca2+-dependent hydrolysis in the forward direction of catalysis, proving direct interaction with Asp351, and yet has little effect on Pi-dependent phosphorylation in the absence of Ca2+, showing that the nucleotide, or at least a portion of it, shifts position with respect to the aspartyl residue upon Ca2+ binding. The phosphorylated aspartate and adjoining residues on the COOH-terminal side, segment 351DKTGTLT357, termed the phosphorylation loop in this study, are highly conserved in P-type ATPases (17Green N.M. Biochem. Soc. Trans. 1989; 17: 970-972Google Scholar), and previous mutational analysis has documented their functional importance (18Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (264) Google Scholar, 19Maruyama K. Clarke D.M. Fujii J. Inesi G. Loo T.W. MacLennan D.H. J. Biol. Chem. 1989; 264: 13038-13042Abstract Full Text PDF PubMed Google Scholar). Besides Asp351, also Lys352, Thr355, Leu356, and Thr357 are critical to Ca2+ transport as well as phosphorylation (19Maruyama K. Clarke D.M. Fujii J. Inesi G. Loo T.W. MacLennan D.H. J. Biol. Chem. 1989; 264: 13038-13042Abstract Full Text PDF PubMed Google Scholar), and further clarification of the distinct roles of these residues in nucleotide binding, phosphoryl transfer, and long range interaction with the Ca2+ sites may aid understanding of energy transduction in the pump. In this study, we assess the effects on nucleotide binding of mutations to the phosphorylation loop residues that previously were shown to result in severely impaired or inactive pumps (18Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (264) Google Scholar, 19Maruyama K. Clarke D.M. Fujii J. Inesi G. Loo T.W. MacLennan D.H. J. Biol. Chem. 1989; 264: 13038-13042Abstract Full Text PDF PubMed Google Scholar). ATP binding is measured mainly through inhibition of TNP-8N3-ATP photolabeling of Lys492, which has recently been successfully applied to Ca2+-ATPase mutated in segment487FSRDRK492 (16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In selected cases where the affinity is extremely high, binding is also measured by a direct filtration assay. In addition, the effects of the mutations on Ca2+ occlusion induced with CrATP are determined. The results show that most of the mutations in the phosphorylation loop affect nucleotide binding and disrupt CrATP-induced Ca2+occlusion. Our analysis of mutations to Asp351 reveals high intrinsic nucleotide binding energies when the negative charge is removed, particularly in the presence of Mg2+ and Ca2+, i.e. in the E1Ca2 state (KD = 0.001 μm for mutant D351A). These favorable interactions may be utilized to gain the transition state and to provoke conformational changes that communicate with the transport sites. The mutant Ca2+-ATPase cDNAs used in this study were the same as those described previously (18Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (264) Google Scholar, 19Maruyama K. Clarke D.M. Fujii J. Inesi G. Loo T.W. MacLennan D.H. J. Biol. Chem. 1989; 264: 13038-13042Abstract Full Text PDF PubMed Google Scholar) but were shuttled to vector pMT2 (20Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W.B. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (333) Google Scholar) to obtain higher expression levels in COS-1 cells (18Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (264) Google Scholar, 21Vilsen B. Andersen J.P. MacLennan D.H. J. Biol. Chem. 1991; 266: 16157-16164Abstract Full Text PDF PubMed Google Scholar). COS-1 cell microsomes containing expressed wild-type or mutated Ca2+-ATPase were isolated by differential centrifugation 48–72 h after transfection (18Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (264) Google Scholar). The exogenous Ca2+-ATPase content of the microsomal fraction was assayed with a specific sandwich enzyme-linked immunosorbent assay (21Vilsen B. Andersen J.P. MacLennan D.H. J. Biol. Chem. 1991; 266: 16157-16164Abstract Full Text PDF PubMed Google Scholar). The synthesis of [γ-32P]TNP-8N3-ATP, photolabeling of COS-1 cell microsomes, the inhibition by ATP, quantification of labeled bands by electronic autoradiography (“imaging”) following SDS-polyacrylamide gel electrophoresis, curve fitting equations and calculations of the “true”KD(ATP) have been described previously (16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 22Seebregts C.J. McIntosh D.B. J. Biol. Chem. 1989; 264: 2043-2052Abstract Full Text PDF PubMed Google Scholar). For fitting of the TNP-8N3-ATP labeling data, the Hill equation with or without a linear component was used, and the Hill coefficient was set to 1. The concentration of free Ca2+was set with 5 mm EGTA and variable amounts of total CaCl2 as calculated according to Fabiato and Fabiato (23Fabiato A. Fabiato F. J. Physiol. 1979; 75: 463-505PubMed Google Scholar) taking the Mg2+ concentration and pH into consideration. CrATP-dependent Ca2+ occlusion was measured as before (16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 24Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar). Equilibrium ATP binding to mutants D351N and D351A was also measured by filtration. COS-1 cell microsomes (1 μl of stock microsomes in 1 ml; approximately 0.5 pmol of Ca2+-ATPase protein/ml) were incubated with [γ-32P]ATP, 1 mm[3H]sucrose, and other components as indicated in the Fig. 7 legend for 1 min at 25 °C, and the sample was filtered on Millipore GS 0.22-μm filters under mild vacuum. The radioactivity of the filter was measured by liquid scintillation counting. The wet volume of the filter was determined from the tritium radioactivity (range: 28–42 μl), allowing determination of the radioactivity of unbound nucleotide, which was subtracted from the total 32P cpm to obtain the amount of ATP bound to the microsomes. The formation of a slowly dissociating CrATP complex with the Ca2+-ATPase in the presence of Ca2+ was followed through the inhibition of [γ-32P]TNP-8N3-ATP photolabeling. Microsomes containing expressed wild-type or mutated Ca2+-ATPase were incubated at 37 °C with CrATP for up to 1 h. Aliquots were taken at timed intervals and diluted 50-fold into irradiation medium with 0.5 μm[γ-32P]TNP-8N3-ATP. The samples were irradiated for 1 min and subjected to SDS-polyacrylamide gel electrophoresis, and the radioactivity was quantified by electronic autoradiography as described previously (16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Twenty-four mutations have previously been introduced into the conserved phosphorylation loop of the Ca2+-ATPase between Ile348 and Thr357 (18Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (264) Google Scholar, 19Maruyama K. Clarke D.M. Fujii J. Inesi G. Loo T.W. MacLennan D.H. J. Biol. Chem. 1989; 264: 13038-13042Abstract Full Text PDF PubMed Google Scholar). All the mutants with alteration to the aspartic acid residue Asp351receiving the phosphoryl group during catalysis are inactive, and so are the mutants with alterations to Lys352, even in the case of the most conservative replacement of Lys352 with arginine. Activity is not affected by conservative replacements of Thr355 or Thr357 with serine, but replacement with alanine reduces the Ca2+ transport activity as well as the level of phosphoenzyme. Replacement of Leu356 with isoleucine is without effect on activity, but mutation to threonine inactivates the pump. Hence, these residues (nine mutants in all; see Table I) were selected for the present study of nucleotide binding properties.Table INucleotide binding parameters and CrATP-dependent Ca2+ occlusion of wild-type Ca2+-ATPase and mutantsK0.5(TNP-8N3-ATP)KD(ATP)1-aThe “true” KD calculated under the assumption of competitive inhibition as described in Ref. 16; the concentration of TNP-8N3-ATP was 3 ×K0.5 except for mutants K352Q and T357A where it was equal to the K0.5.Ca2+occlusion1-b−, no occlusion; +, partial occlusion; ++, full occlusion; ND, not determined. Data are shown in Fig. 2.μmμmWild type0.980.54++D351N0.00630.0065−D351T0.0530.012NDD351A0.0400.025−D351E1.30.88NDK352R1.07.1+K352Q4.9∼500−T355A0.740.93+L356T>50 or no specific labeling−T357A6.4∼20−For nucleotide binding, medium was 25 mm HEPPS/TMAH, pH 8.5, 20% (w/v) glycerol, 1 mm MgCl2, 0.5 mm EGTA. For CrATP-dependent Ca2+occlusion, medium was 50 mm TES/Tris, pH 7.0, 100 mm NaCl, 5 mm MgCl2, 40 μm45CaCl2, and 1 mm CrATP.1-a The “true” KD calculated under the assumption of competitive inhibition as described in Ref. 16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar; the concentration of TNP-8N3-ATP was 3 ×K0.5 except for mutants K352Q and T357A where it was equal to the K0.5.1-b −, no occlusion; +, partial occlusion; ++, full occlusion; ND, not determined. Data are shown in Fig. 2. Open table in a new tab For nucleotide binding, medium was 25 mm HEPPS/TMAH, pH 8.5, 20% (w/v) glycerol, 1 mm MgCl2, 0.5 mm EGTA. For CrATP-dependent Ca2+occlusion, medium was 50 mm TES/Tris, pH 7.0, 100 mm NaCl, 5 mm MgCl2, 40 μm45CaCl2, and 1 mm CrATP. The assay for nucleotide binding, which is based on specific [γ-32P]TNP-8N3-ATP photolabeling of Lys492 and nucleotide competition, has been validated previously (16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Results obtained under optimum labeling conditions at pH 8.5 demonstrated that this assay is able to produce highly accurate values for TNP-8N3-ATP and ATP binding affinities of Ca2+-ATPase expressed in COS-1 cell microsomes (16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In assessing the results to be described below, it is furthermore useful to know that TNP-8N3-ATP is a substrate of the Ca2+-ATPase, albeit a slow one, whether untethered or tethered to Lys492 by photolabeling (15McIntosh D.B. Woolley D.G. J. Biol. Chem. 1994; 269: 21587-21595Abstract Full Text PDF PubMed Google Scholar, 16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). This means that the position of the γ-phosphoryl group of the bound nucleotide must be similar, although probably not identical, to that of bound ATP. The concentration dependence of TNP-8N3-ATP photolabeling of wild-type and mutant Ca2+-ATPases at pH 8.5 in the presence of Mg2+ and absence of Ca2+ (presence of EGTA) is shown in Fig. 1 A. The data could be fitted satisfactorily to the sum of a simple hyperbolic binding function and a linear component, the latter representing nonspecific labeling as previously explained (16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). For most of the mutants, the linear component was small and insignificant, but as seen in Fig. 1 the linear component was rather prominent for mutant K352Q, for unknown reasons. The derived K0.5 values corresponding to the hyperbolic component are listed in Table I. It can been seen that removal of the negative charge on Asp351, as shown by mutants D351N, D351A, and D351T, led to a pronounced increase in TNP-8N3-ATP affinity, with D351N exhibiting the largest increase of 156-fold. By contrast, mutation D351E, which conserves the negative charge, did not significantly affect the TNP-8N3-ATP binding affinity. The concentration of Ca2+-ATPase in the irradiation assay was approximately 0.4 nm for the tightly binding mutants and approximately 2 nm for the rest to ensure a reasonably high ratio of free to bound nucleotide, thereby allowing the total concentration to be equated with the free concentration. Mutation K352Q, which removes the positive charge of Lys352, lowered the affinity for TNP-8N3-ATP at least 5-fold, whereas the more conservative replacement with arginine, K352R, was without significant effect. Mutation T355A was likewise silent, but T357A lowered the affinity for the TNP nucleotide about 6-fold. Mutant L356T exhibited a low level of labeling that was linear with increasing concentrations of TNP-8N3-ATP up to 30 μm. This indicates that either Lys492 was not being labeled or the affinity was extremely poor (K0.5 estimated to be >50 μm). The inhibition of photolabeling by ATP under the same buffer conditions is shown in Fig. 1 B, and the derived true KD values assuming competitive inhibition are listed in Table I. Usually, the concentration of TNP-8N3-ATP was fixed at 3 × K0.5 to ensure that the binding site is close to saturation; however, in the case of mutants K352Q and T357A, inhibition was so poor at these concentrations that the TNP-8N3-ATP concentrations were lowered to the K0.5 in each case. It is apparent that removal of the negative charge on Asp351 caused a large increase in affinity for ATP (83-fold, 45-fold, and 22-fold for D351N, D351T, and D351A, respectively) similar to that seen for TNP-8N3-ATP. Mutations D351E and T355A appeared to slightly decrease the affinity for ATP (1.5–2-fold compared with wild type). While mutation K352R resulted in a 13-fold reduction of ATP affinity, K352Q led to a spectacular effect, reducing the ATP affinity close to 1000-fold. Mutation T357A decreased the affinity for ATP at least 40-fold. Because the affinity of the latter two mutants was too low for complete inhibition to be reached, the choice of the offset of the binding curve was somewhat uncertain in these cases, resulting in corresponding uncertainties with respect to the exact KD(ATP) values. Hence, the results shown in Fig. 1 indicate that the most disruptive mutation in terms of nucleotide binding is L356T, followed by K352Q, T357A, and K352R. The latter three mutations affected ATP binding much more than the binding of TNP-nucleotide, similar to the situation with mutations close to Lys492 (16McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). All of the mutants in which the negative charge on Asp351 was removed exhibited a large increase in affinity for both nucleotides, with D351N being the most dramatic. On the other hand, mutation D351E had little effect on the binding of either nucleotide. Nucleotide binding and the coupling between the catalytic and transport sites can be assessed by measuring CrATP-induced Ca2+occlusion. CrATP slowly forms a complex with the Ca2+-ATPase at the catalytic site without phosphorylating Asp351 and causes simultaneous occlusion of Ca2+ at the transport sites (24Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar, 25Serpersu E.H. Kirch U. Schoner W. Eur. J. Biochem. 1982; 122: 347-354Crossref PubMed Scopus (51) Google Scholar, 26Vilsen B. Andersen J.P. Bochim. Biophys. Acta. 1986; 855: 429-431Crossref PubMed Scopus (38) Google Scholar, 27Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 3539-3550Abstract Full Text PDF PubMed Google Scholar). Because this complex is very stable, requiring hours to dissociate,45Ca2+ occlusion with CrATP can be measured in Ca2+-ATPase expressed in COS-1 cell microsomes by size exclusion HPLC following detergent solubilization of the microsomes (24Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar). The results of such measurements on the wild type and selected mutants are shown in Fig. 2 and summarized in Table I. The microsomes were incubated 1 h at 37 °C with 1 mm CrATP and 40 μm45Ca2+, i.e. just about enough to ensure saturation of the occlusion reaction in the wild-type enzyme, before solubilization and chromatography. Equal amounts of expressed Ca2+-ATPase, according to enzyme-linked immunosorbent assay measurements, were chromatographed, so the elution profiles are comparable. The control represents microsomes without expressed Ca2+-ATPase, i.e. harvested from COS-1 cells mock-transfected with the expression vector without insert. The amount of control microsomes in mg of total membrane protein corresponds to that chromatographed in the case of the wild type. The distinct peak of radioactivity eluting between 14 and 15 min in the experiment with the wild type and in some of the experiments with mutants represents Ca2+ occluded in the detergent-solubilized monomeric enzyme (24Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar). As can be seen, there was no such peak for mutants D351N and D351A, and hence these mutants did not occlude Ca2+. Mutants K352R and T355A showed partial occlusion, and mutants K352Q, L356T, and T357A also failed to occlude Ca2+. Thus, for some mutations, notably those that slightly or grossly lower the affinity for ATP, the effect on CrATP-induced Ca2+occlusion seems to be correlated with the change in ATP binding affinity. However, mutations to Asp351, which caused a huge increase in affinity for ATP, also appeared to prevent CrATP-induced Ca2+ occlusion. In order to elucidate whether this was due to defective CrATP complexation at the catalytic site or an uncoupling of CrATP complexation and Ca2+ occlusion, we devised an assay wherein the microsomes were incubated with CrATP for up to 1 h at 37 °C and then diluted substantially prior to photolabeling with TNP-8N3-ATP. The affinity of the Ca2+-ATPase for CrATP is not high, and a concentration of CrATP in the millimolar range is required to obtain saturation so that occlusion occurs in a reasonable period of time. To minimize the competitive binding of contaminant ATP that might be present in the CrATP preparation, the samples were irradiated under conditions where the affinity for TNP-8N3-ATP is reasonably high and that for ATP is fairly low (pH 8.5 in the presence of EDTA). Also with this in mind, a concentration of TNP-8N3-ATP of approximately 10× K0.5 was chosen. As seen in Fig.3, photolabeling of the wild-type Ca2+-ATPase was inhibited by CrATP in a time-dependent manner, indicative of a gradual and effectively irreversible complexation of CrATP at the catalytic site. In mutants D351N and D351A, CrATP failed to inhibit photolabeling, showing that the irreversible binding of CrATP had not occurred. This explains the lack of CrATP-induced Ca2+ occlusion in these mutants. The buffer conditions in the above binding experiments with TNP-8N3-ATP and ATP (pH 8.5, in the presence of Mg2+ and absence of Ca2+) largely favor accumulation of the E1 state of the wild-type Ca2+-ATPase, whereas the E2H and E2H3 forms (cf. Scheme 1) would predominate at neutral and acid pH in the absence of Ca2+ (7Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar). To better understand the huge increase in nucleotide affinity induced by mutations D351N and D351A and the implications for the catalytic mechanism, we investigated the influence of pH, Mg2+, and Ca2+, as well as thapsigargin, a tightly binding inhibitor that appears to lock the enzyme into E2 or an “E2-like” state (28Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1991; 267: 1286-1292Abstract Full Text PDF Google Scholar). Results of these studies are presented in Figs.Figure 4, Figure 5, Figure 6and summarized in Table II in terms of derived dissociation constants.Figure 5Effect of Mg2+ and Ca2+ on TNP-8N3-ATP photolabeling (A and B) and ATP inhibition (C and D) of mutant D351N at pH 7.0 (A and C) and pH 8.5 (B and D). Photolabeling was performed as in Figs. 1 and4, in the presence of 1 mm MgCl2 + 0.5 mm EGTA (○), 2 mm EDTA (▵), or 1 mm MgCl2 plus 0.05 mmCaCl2 (■).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Effect of Mg2+ and Ca2+ on TNP-8N3-ATP photolabeling (A and B) and ATP inhibition (C and D) of mutant D351A at pH 7.0 (A and C) and pH 8.5 (B and D). Photolabeling was performed as in Figs. 1 and4, in the presence of 1 mm MgCl2 plus 0.5 mm EGTA (○), 2 mm EDTA (▵), or 1 mm MgCl2 plus 0.05 mmCaCl2 (■).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIEffect of Mg2+, Ca2+, pH, and thapsigargin on nucleotide binding parameters of wild-type Ca2+-ATPase and mutants D351N and D351AConditionsCa2+-ATPaseK0.5(TNP-8N3-ATPKD(ATP)μmμmpH 8.5 MgCl2, EGTA2-aStandard medium: 25 mm buffer, 20% (w/v) glycerol, 1 mm MgCl2, 0.5 mm EGTA.WT2-bWT, wild type.0.980.54D351N0.00630.0065D351A0.0400.025 Thapsigargin2-cStandard medium with 0.01–0.09 μmthapsigargin (approximately 10 mol of thapsigargin/mol of Ca2+-ATPase).WT1.2195D351N0.0965.0D351A0.4721 CaCl22-dMedium: 25 mm buffer, 20% (w/v) glycerol, 1 mm MgCl2, 50 μm CaCl2.D351N0.00580.028D351A0.00350.0011 EDTA2-eMedium: 25 mm buffer, 20% (w/v) glycerol, 2 mm EDTA.WT0.1118D351N0.0141.3D351A0.0501.1pH 7.0 MgCl2, EGTA2-aStandard medium: 25 mm buffer, 20% (w/v) glycerol, 1 mm MgCl2, 0.5 mm EGTA.WT0.488.4D351N0.0600.57D351A0.21

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