Roles of Conserved P Domain Residues and Mg2+ in ATP Binding in the Ground and Ca2+-activated States of Sarcoplasmic Reticulum Ca2+-ATPase

Residues in conserved motifs 625TGD, 676FARXXPXXK, and 701TGDGVND in domain P of sarcoplasmic reticulum Ca2+-ATPase, as well as in motifs 601DPPR and 359NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg2+ binding and stabilization of the Ca2+-activated transition state for phosphoryl transfer. In the absence of Mg2+, mutations removing the charges of domain P residues Asp627, Lys684, Asp703, and Asp707 increased the affinity for ATP and 2′,3′-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5′-triphosphate. These mutations, as well as Gly626→ Ala, were inhibitory for ATP binding in the presence of Mg2+ and for tight binding of the β,γ-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg2+. The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg2+ is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly626 → Ala, Lys684 → Met, Asp703 → Ala/Ser/Cys, and mutants with alteration to Asp707 exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg2+ and Ca2+. Under these conditions, ATP binding was almost completely blocked in Gly626 → Ala and occurred with 12- and 7-fold reduced affinities in Asp703 → Ala and Asp707 → Cys, respectively, relative to the situation in the presence of Mg2+ without Ca2+, whereas in Lys684 → Met and Asp707 → Ser/Asn the affinity was enhanced 14- and 3–5-fold, respectively. Hence, Gly626 and Asp703 seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca2+ binding at the transport sites. Residues in conserved motifs 625TGD, 676FARXXPXXK, and 701TGDGVND in domain P of sarcoplasmic reticulum Ca2+-ATPase, as well as in motifs 601DPPR and 359NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg2+ binding and stabilization of the Ca2+-activated transition state for phosphoryl transfer. In the absence of Mg2+, mutations removing the charges of domain P residues Asp627, Lys684, Asp703, and Asp707 increased the affinity for ATP and 2′,3′-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5′-triphosphate. These mutations, as well as Gly626→ Ala, were inhibitory for ATP binding in the presence of Mg2+ and for tight binding of the β,γ-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg2+. The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg2+ is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly626 → Ala, Lys684 → Met, Asp703 → Ala/Ser/Cys, and mutants with alteration to Asp707 exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg2+ and Ca2+. Under these conditions, ATP binding was almost completely blocked in Gly626 → Ala and occurred with 12- and 7-fold reduced affinities in Asp703 → Ala and Asp707 → Cys, respectively, relative to the situation in the presence of Mg2+ without Ca2+, whereas in Lys684 → Met and Asp707 → Ser/Asn the affinity was enhanced 14- and 3–5-fold, respectively. Hence, Gly626 and Asp703 seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca2+ binding at the transport sites. The sarcoplasmic reticulum Ca2+-ATPase 1The abbreviations used are: Ca2+-ATPase, the sarco(endo)plasmic reticulum Ca2+-transporting adenosine triphosphatase (EC 3.6.1.38); CrATP, β,γ-bidentate chromium(III) complex of ATP; E1, enzyme form with cytoplasmically facing high affinity Ca2+ binding sites; E2, enzyme form with low affinity for Ca2+; E2(TG), E2 enzyme with bound thapsigargin; E1P, ADP-sensitive phosphoenzyme intermediate containing Ca2+ in the occluded state; E2P, ADP-insensitive phosphoenzyme intermediate with luminally facing low affinity Ca2+ binding sites; EPPS, N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid; K0.5, ligand concentration giving half-maximum effect; MES, 2-[N-morpholino]eth-anesulfonic acid; MOPS, 3-[N-morpholino]propanesulfonic acid; TMAH, tetramethylammonium hydroxide; TNP-8N3-ATP, 2′,3′-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5′-triphosphate. from rabbit muscle couples the hydrolysis of ATP to the transport of Ca2+ to the reticular lumen, and the lowering of the sarcoplasmic Ca2+ concentration leads to muscle relaxation. This ion pump is a well studied example of P-type ATPases, the catalytic cycle of which includes a phosphorylated aspartyl intermediate and progression through E1 and E2 states. ATP-dependent phosphorylation takes place upon Ca2+ binding in the E1 form, and hydrolysis of the aspartyl phosphoryl bond occurs in the E2P phosphoenzyme conformation following Ca2+ translocation across the membrane. Two atomic structures of the protein have been elucidated by x-ray crystallography, one in the presence of Ca2+ (designated E1(Ca2)) and another in the absence of Ca2+ and presence of the specific inhibitor thapsigargin (E2(TG)) (1Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Google Scholar, 2Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Google Scholar). They reveal 10 membrane-spanning helices, connected through a stalk section to the phosphorylation (P) domain in association with nucleotide (N) and actuator (A) domains. The three head domains are loosely attached in E1(Ca2) and closer together, forming a more compact entity, in E2(TG). For ATP-dependent phosphorylation of Asp351 to occur in the Ca2+ bound E1 state, domain N must close over the phosphorylation site, such that the nucleotide site is brought close to Asp351. ATP likely straddles both domains, the nucleoside portion anchored in domain N and the phosphates stretching into domain P (cf.Fig. 1A). Domain P contains the highly conserved segments 625TGD, 676FARXXPXXK, and 701TGDGVND that surround the phosphorylation loop 351DKTGTLT. Many of these residues have been implicated in ATP binding and catalysis on the basis of chemical labeling and cross-linking experiments, and mutagenesis of Ca2+-ATPase and related pumps (for review, see Ref. 3McIntosh D.B. Adv. Mol. Cell. Scholar). of at the site of a of and to have a Scholar). structures of intermediate or have a of the in the of and show of the site and to a tight transition state the phosphoryl group or Ref. H. Mol. 2002; and The residues in E1(Ca2) and structures of Ca2+-ATPase are with of and the or the group or is required in the ion pump to the transition state for phosphoryl transfer. Hence, an is to reveal such in the Mg2+, to structures of and in is the phosphorylation loop Asp703 in and the phosphoryl group (cf.Fig. a of the ATP the of E1(Ca2) were in the presence of high Mg2+ and Ca2+, and was at the catalytic Mg2+ site of the absence of ATP. Ca2+ binding at the transport phosphoryl transfer to Asp351 and another required for the to the transition The on of domain P mutations, which the of the conserved phosphorylation loop in binding D.B. Scholar, D.B. Scholar). on the conserved segments of the P domain have mutations of of the conserved residues in these the Mg2+ binding Asp703, as well as Lys684 and and studied mutants Scholar, Scholar, for of nucleotide binding and of phosphorylation, to assess the roles of these residues in nucleotide and Mg2+ binding and stabilization of the Ca2+-activated transition state for phosphoryl transfer. Residues in the highly conserved hinge 601DPPR and which the N and P domains, have been studied to and the of the of the mutants exhibited very slow or negligible phosphorylation, it was possible to measure ATP binding in the presence of both Mg2+ and Ca2+, in the with ATP in the transition state for phosphoryl transfer. and mutagenesis of the rabbit muscle Ca2+-ATPase was as Scholar). For the in Mol. Cell. was into by C. H. Mol. Cell. Scholar). The containing or Ca2+-ATPase was by Scholar). The concentration of protein was by a specific Scholar). at were a as 2000; Scholar). The was by the of ATP to the phosphorylation and were by D.B. Scholar). was with of containing The protein was by and to in a at and the with the Ca2+-ATPase was by a The phosphorylation was in with from for of the of containing or Ca2+-ATPase the by and the of by following were as D.B. Scholar). The concentration of was in the with ATP. of the Ca2+-ATPase with at and the of enzyme with by with in following a as D.B. and in the to the was as by and in Scholar). and phosphorylation data and the labeling data were as 2000; Scholar, D.B. Scholar). The for ATP and binding was the for of the labeling D.B. Scholar). The mutants examined are in the it is to the of these residues in the phosphorylation site and ATP and Mg2+ on the of Ca2+-ATPase in the conformation C. Nomura H. Nature. 2002; 418: 605-611Google the atomic of the with bound H. Mol. 2002; and an of ATP bound to domain N of M. is in ATP is in The portion of ATP is in domain and the and in domain P in a close to the the from ATP. The ATP is the and by to both and it is that of the domains is for the ATP to both binding sites. This is more in the E1(Ca2) Ca2+-ATPase (1Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Google which to the E1(Ca2) for the ATP binding site. In a transition of is bound as a phosphoryl transition state Mg2+ is by a the residues to Asp351 and Asp703, the of the of and is to of residues to and as well as to of the of and Lys684 H. Mol. 2002; and Hence, these residues are as of Mg2+ and respectively, in the transition state of the binding and phosphorylation in of in of in of were as for and The for Lys684 → was from Ref. of at was by with ATP and as for of was on basis of the data in P → → → → is on the data in Ref. → that the data are from → → → → → → → → → → → of of of were as for and The for Lys684 → was from Ref. 2000; at was by with ATP and as for was on basis of the data in that the data are from is on the data in Ref. in a with and without Mg2+ in the of of ATP binding to Ca2+-ATPase in is possible by of the low of have a binding on specific of in domain N and by for binding D.B. Scholar). the concentration of a which is the concentration at which is and the ATP concentration of of is studied at a concentration of to This a for ATP binding from which the for ATP binding is the for is a for the Ca2+-ATPase both bound and attached to the hydrolysis is slow for D.B. Scholar). The binding with and the mutants are in and of the data are in In the of and more from are in of an in affinity are in For to of the a reduced affinity is upon For charged of the by to an of the affinity for the is electrostatic in the domain P mutations in the absence of Mg2+ it that mutations increased the affinity for and ATP. This was for the affinity was increased and respectively. the affinity for which as an that the group is the electrostatic environment as the their in related but in with been for Asp351 D.B. of and Asp703 with the had on binding of or are that in of the residues to in of the phosphates of or ATP in the absence of Mg2+, and electrostatic In the presence of Mg2+, a effects on and Under the of ATP in the form of the with a This complex to enzyme with affinity whereas the Mg2+ complex of with affinity the nucleotide to the mutations increased the affinity for had whereas the relative to The in affinity for Met is particularly in affinity were for mutations of as to the situation in the absence of Mg2+. of Asp703 Mg2+, the affinity for more in the Asp703 mutants in the Hence, Lys684, and Asp703 to of the Mg2+ binding with In domain P mutations the affinity for The critical residues seem to Gly626 and Lys684, for which with and respectively, to and of that the of Gly626 and group of Lys684 roles in the of In the of the of affinity were for mutations removing the is to in these is the affinities for ATP and the affinity for ATP is increased and the affinity for with for of or Asp703 to had on the affinity for with the was and the is and are on the hinge segments domains N and P (cf.Fig. 1A). of these residues had or on ATP binding in the absence of Mg2+, but in the presence of Mg2+, Ala and in affinity for both whereas was from and the presence of Ca2+, the with leads to phosphoryl transfer to Asp351 with of the ADP-sensitive phosphoenzyme was that Gly626→ Ala, Ala, and are to form of phosphoenzyme upon with and Ca2+ for 10 at whereas and form a phosphoenzyme these Scholar, Scholar, Scholar). were to these for which phosphorylated to a in to mutants that have been of phosphoenzyme in the of Ala and whereas Met, Cys, Cys, and were to form a of phosphoenzyme with and Ca2+ at to the of phosphorylation at a 2000; Scholar). the Met and the three mutants with alteration to were of phosphorylation from in the presence of Mg2+ and Ca2+ → of data these are in and and the are in the concentration is close to the for phosphorylation of in the affinity for as well as the for phosphorylation are in the phosphorylation In the hinge Ala had on the phosphorylation whereas both mutations of of the phosphorylation at was for mutations of Gly626→ Ala and of Asp703 to or reduced the of phosphorylation and the data for whereas the more of Asp703 to and only reduced the phosphorylation to and respectively, with and is that phosphoenzyme in the low of phosphorylation, that a in the to is This was examined for Ala, as in the upon of the phosphoenzyme with ATP was on the upon of to the phosphorylation that the phosphoenzyme intermediate was ADP-sensitive and that the slow in the is the → E2P the mutants only Ala and Ca2+ transport of in the presence of a high concentration of at and data as in Ref. D.B. Scholar). or of the → E2P transition been for mutants and on the basis of to in Scholar, Scholar). these a of the → E2P transition in and and a in Ala and and → E2P is the in the transport cycle at ATP and Ca2+ the of must a reduced phosphorylation to the Ca2+ transport in the in the of the presence of both Mg2+ and Ca2+, is a of the Ca2+-ATPase D.B. Scholar). Hence, it is possible to measure or ATP binding by the these conditions, the group both from and from the ATP used to the for the mutants or very low phosphorylation the with both Mg2+ and Ca2+ the the transition state in the phosphoryl transfer are in and to a Ca2+ was only for Met, the binding affinity was relative to the situation without Ca2+. were in affinity for the Asp703 and Asp707 the in affinity for In the of the were and for of the In the the of Ca2+. In Gly626→ Ala, the ATP affinity was very low in the presence of Ca2+, reduced relative to the situation with Mg2+ Ca2+ the ATP affinity in In Ca2+ increased the ATP affinity in and in ATP affinity were for and was the Ca2+ lowering the affinity Ca2+ had on In Cys, Ca2+ a which is to that in these the Mg2+ concentration was the Ca2+ that the of the nucleotide was as the Mg2+ Hence, the by Ca2+ are by Ca2+ binding at the transport sites. of roles of these residues in nucleotide binding were by the at which bound at the site in the presence of Ca2+ Scholar, Scholar). have that it is possible to the of tight binding in an in which the enzyme is with at for by into containing TNP-8N3-ATP, which the enzyme bound D.B. Scholar). are in and in of the following for in the absence and presence of In the presence of CrATP, the of the to the of the complex with place with a of for whereas in the absence of the over the For Gly626 → Ala, was of CrATP, that tight complex with was Asp703 → and Asp707 → of CrATP, but the of the tight complex was in these mutants with In the the mutations of the of the tight and Ala was For Met, and a in labeling with was in the absence of The presence of of these mutants to a tight complex with Mg2+ and of from in Asp703 → the presence of Mg2+, but without Ca2+, the Ca2+-ATPase in E2 conformation phosphorylated by in a the of E2P in the transport the mutants examined in the only Ala, and were phosphorylated by that are in with and Mg2+ for 10 at in the presence of with and are in For the affinities for and Mg2+ and with is in that the affinity for is reduced relative to whereas the affinity for Mg2+ is reduced as as that the of a group to the of Asp703 a of the with Mg2+ in the E2 For an of the Mg2+ concentration to as as of a of In the have that Mg2+ and Ca2+ at the catalytic site and transport have effects on the of the Ca2+-ATPase with and residues in these and of Mg2+ in was effects on nucleotide binding in the absence and presence of Mg2+. In the absence of Mg2+, mutations removing the charges of domain P residues Asp627, Lys684, Asp703, and Asp707 enhanced and ATP of the to these residues in the but In the presence of Mg2+, these mutations, as well as Gly626→ Ala, were inhibitory for ATP that in the is close of these residues with For TNP-8N3-ATP, only mutations of the loop were inhibitory in the presence of Mg2+, the of the at from the P domain residues these of the hinge and had effects on ATP binding in the presence of Mg2+, and only effects on binding in the absence of Mg2+. the basis of the atomic of in a transition H. Mol. 2002; and domain P of Ca2+-ATPase must from the E1(Ca2) and atomic structures for phosphorylation to Hence, the the of and the of Asp703 and and that the of Gly626 and the group of Lys684 to by are to the transition state of the in 1A). Asp351. The is from the of the group of and to for an in and for of Mg2+, as in the transition state of the and of the in these residues closer is the electrostatic three Asp703, and surround one the of the phosphorylation well are and The of in the the electrostatic of these conserved domain P residues the of electrostatic effects a of the on one is bound to and Lys684 is Asp351 and Asp707 in the and the on the to the of the that one and the the on one of the the to the In with are the effects of of Lys684 or The data with for mutations of the phosphorylation D.B. Scholar, D.B. that the of conserved domain P residues is a of ATP and Mg2+ and that Ca2+ binding at the transport to the tight in the transition Mg2+ and Ca2+ the to in domain P, as the structures show (1Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Google Scholar, 2Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Google Scholar). ATP to electrostatic as with the of a electrostatic at the phosphorylation site. The electrostatic effects on ATP binding in the absence of Mg2+ and Ca2+ are with more in affinity for mutations D.B. and The with the was for the nucleotide Met increased the affinity for both ATP and and the of of Asp351 and Asp707 as effects seem to to Asp627, a of the phosphorylation well a of affinity was upon of the in the absence of Mg2+, the of both and ATP to into of these but effects The phosphates likely at and in domain to D.B. and at stretching from domain P D.B. Scholar). Mg2+ for ATP binding in domain P, the mutations were in the presence of Mg2+ and absence of Ca2+, from and mutations of the to of Lys684 with was for binding in the absence of Ca2+ of the with Lys684 the group in the Ca2+-ATPase as in (cf.Fig. is the of binding by mutations of Asp703, which is a Mg2+ on the basis of the H. Mol. 2002; and In the absence of Ca2+, affinity was reduced by of Asp703 with or whereas ATP affinity was the with a in affinity to that with it that the negatively charged group of the Asp703 is required for in the absence of Ca2+. of Asp703 with in of or in the E1 form, which is with the relative of in the atomic structures and to the in the The phosphorylation of the E2 form with was very to mutations of Asp703 and the with a of the affinity for Mg2+ as of phosphorylation by with a critical of Asp703 in Mg2+ in the E2P phosphoenzyme For and of relative to were in the absence of Ca2+. In these as well as of the Asp703 the increased affinity for ATP and the reduced affinity for into or the affinities for ATP and Hence, the binding data the roles of Asp703, and in In three of by the of from the of the In is by (cf.Fig. for the in P-type to domains N and P together, and at the with the of bound ATP (cf.Fig. 1A). In the to Asp707 is to a Mg2+ to the in a conformation the of Asp703 to Mg2+ (cf.Fig. in Ca2+-ATPase with the of in binding affinity for Asp707 and Asp703 mutations removing the In the of mutations enhanced which only that electrostatic effects to but that Asp707 to of the Mg2+ ion of Met enhanced in with the that the of nucleotide into the of Asp703 the binding of in to the situation with Mg2+ anchor the of TNP-8N3-ATP, the that these are bound at D.B. Scholar). CrATP, on the to the P domain mutations to binding or of the tight enzyme complex with the chromium(III) ion takes a to that of the Mg2+ of with the phosphates into the phosphorylation This with that of Asp351 tight binding D.B. Scholar). that the group of is to Asp351 in the the as the Ca2+ bound in an occluded state at the transport Scholar, that with chromium(III) Mg2+ the transition state for phosphoryl transfer The tight binding of likely from of one or more of the with the chromium(III) ion and the and with or of the from Asp351 D.B. from of the P domain residues studied The Ca2+-activated negligible phosphorylation of of the mutants an to the in the pseudo-transition state in the presence of both Mg2+ and Ca2+. Ca2+ binding and in affinity for of the with a closer of the residues the phosphates and Mg2+. that which is in phosphorylation, a very high affinity for ATP in the presence of Mg2+ and the of Ca2+ the affinity D.B. Scholar). For Ala, the is the affinity is high with Mg2+ and with both Ca2+ and Mg2+ D.B. Scholar). These are for is at the phosphorylation site in the transition state and for of the the group of the is with in the state, but the is more in the tight in have effects on binding in a transition the Asp351 the phosphoryl group from to required for the and with in stabilization of the transition state for phosphoryl a of nucleotide affinity occurred upon Ca2+ binding in the Ala likely that a of with occurs in the transition state in of the of phosphoryl transfer in the Ca2+ is to the binding affinity in mutants with to residues in with the nucleotide in the transition In the of Met, the affinity for increased on the of Ca2+, which is to the for Ala the affinities for are very in the Asp351 and Lys684 both a affinity upon Ca2+ and of these required for the with nucleotide by Ca2+ in the transition the and mutants only and of the affinity for respectively, on the of Ca2+, but with of Asp707 in transition state The of Asp707 is by the 7-fold of affinity for upon Ca2+ of affinity was upon of Ca2+ in Ala, that the presence of Asp703 is required for the of affinity Hence, Asp703 to a more critical in binding in the transition state for phosphoryl transfer in the presence of Ca2+ in the enzyme complex with in the absence of Ca2+. In the Ca2+ binding at the transport very tight Mg2+ by Asp703 in the presence of the phosphoryl transfer. the of Asp703 with only phosphorylation, in to the almost of phosphorylation for Ala/Ser/Cys, that a group for in the Ca2+-activated likely that in the transition state the of Asp703 only one to Mg2+ as in (cf.Fig. The of Gly626→ Ala are in the effects of Mg2+ and Ca2+ on The of the of of is by electrostatic and the nucleotide binding in the absence of Mg2+, or with the that the is in the phosphorylation well these of Mg2+ the to in affinity for and more is the that Ca2+ binding at the transport ATP binding to the Gly626→ Ala in with the in and the transition state in data are with the with the of Gly626 in the presence of Mg2+ and Ca2+. Ca2+ binding in the presence of nucleotide a in the binding site in the of Gly626 that is by of the E1(Ca2) and In it is to that of a with site to Ca2+-ATPase, a with the to to the or Scholar). of P Domain Residues in → E2P is that of of the charged residues in domain P that were for or binding the → E2P transition This that the unfavorable electrostatic environment in domain P to a in the of and of conformation the to E2P of domain N and in a In with the of the → E2P transition by binding of ATP with low affinity to the phosphoenzyme the of unfavorable electrostatic with an group into the phosphorylated catalytic site in the of D.B. Scholar). The effects on the → E2P transition related to in Mg2+ as Mg2+ binding is required for a of the → E2P transition M. Scholar). the the Ala and mutants is the a of → E2P transition the that in the as in the transition state for phosphoryl a group for the The and are in the hinge segments and are at the of on of the catalytic cycle in which such are critical and that the binding of or ATP was only with Mg2+ that Mg2+ is required for of domain N over domain This very well with Mg2+ of the with critical domain P mutations → Ala and → enhanced that to binding in the in hinge The for of domain N and following phosphorylation, which with the reduced of the → E2P transition in these was for both and ATP binding in the presence of Mg2+, as well as for the → E2P that at the of the hinge the domains in with nucleotide binding and in the their effects on both mutations of the of phosphorylation from and Ala had on phosphorylation, the increased affinity for in to the that of domains N and P that in the of the is required to the transition state in the phosphorylation have the roles of catalytic Mg2+ and conserved P domain and hinge residues in the of ATP. Ca2+ binding in the of Gly626 and Asp703 with and in domain P seem of the for mediating entry into the transition state for phosphoryl transfer from ATP. The in domain P to a of Mg2+, and Ca2+ of binding of the to the transition state at the site is to P-type as transport and to from the the site in the domain are and to binding at transport sites. The that in the Ca2+-ATPase the close of Gly626 and Asp703 with on Ca2+ binding at the transport demonstrate an from which to ion binding to phosphoryl transfer in the transport and of and and of for are to of for on

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