The binding of ATP and magnesium to the calcium adenosinetriphosphatase of sarcoplasmic reticulum follows a random mechanism

Reinstein, Jochen; Jencks, William P.

doi:10.1021/bi00077a016

Cited by 16 publications

(13 citation statements)

References 49 publications

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“…Rather, these results support earlier suggestions regarding the presence of a flexible hinge sensitive to calcium activation (46). Furthermore, the extent of conformational heterogeneity in both the calcium-bound and calcium-free conformations is consistent with previous kinetic measurements demonstrating that calcium activation does not significantly affect the kinetics of ATP association with the Ca-ATPase (54,55). Likewise, ATP binding has essentially no effect on the kinetics of calcium association or the dynamics of the N domain (46,56).…”

Section: Discussionsupporting

confidence: 92%

Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity between Nucleotide Binding and Phosphorylation Domains

2004

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High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 degrees involving the nucleotide-binding (N) domain is required to permit the transfer of the gamma-phosphoryl group of ATP to Asp(351) in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientations of the N domain relative to the P domain in the different crystal structures of the Ca-ATPase following calcium activation and the structures of other P-type ATPases suggests the presence of conformational heterogeneity in solution, which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer to measure the spatial separation and conformational heterogeneity between donor (i.e., 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid) and acceptor (i.e., fluorescein 5-isothiocyanate) chromophores covalently bound to the P and N domains, respectively, within the Ca-ATPase stabilized in different enzymatic states associated with the transport cycle. In comparison to the unliganded enzyme, the spatial separation and conformational heterogeneity between these domains are unaffected by enzyme phosphorylation. However, calcium activation results in a 3.4 A increase in the average spatial separation, from 29.4 to 32.8 A, in good agreement with the 4.3 A increase in the distance estimated from high-resolution structures where these sites are respectively separated by 31.6 A (1IWO.pdb) and 35.9 A (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. These results suggest that the approximation of cytoplasmic domains accompanying calcium transport, as observed from crystal structures, occurs in solution within the context of large amplitude domain motions important for catalysis. We suggest that these domain motions enhance the rates of substrate (ATP) access and product (ADP) egress and the probability of a productive juxtaposition of the gamma-phosphoryl moiety of ATP with Asp(351) on the phosphorylation domain to facilitate enzyme phosphorylation and calcium transport.

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Section: Discussionsupporting

confidence: 92%

Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity between Nucleotide Binding and Phosphorylation Domains

2004

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“…The effects of Mg 2ϩ were also quite different for the two nucleotides, the divalent cation strongly enhancing ATP binding (33-fold at pH 8.5 and 9-fold at pH 7.0) but diminishing the affinity for TNP-8N 3 -ATP, especially at pH 8.5. A trend to higher affinity for ATP in the presence of Mg 2ϩ and at alkaline pH has previously been reported for the Ca 2ϩ -ATPase in the native sarcoplasmic reticulum membrane (9,29,30), although the effects appear to be somewhat larger in the present study, possibly because of the presence of glycerol (31), which is added as a nitrene scavenger.…”

Section: Atp Binding and The Phosphorylation Loop Of Sr Ca 2ϩ -Atpasesupporting

confidence: 54%

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

McIntosh¹,

Woolley²,

MacLennan³

et al. 1999

Journal of Biological Chemistry

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-and pH-dependent equilibrium (7) of several (E1/E2) conformational states (Scheme 1) that appear to interact differently with ATP. E2H 3 7 E2H 7 E1 7 E1Ca 7 E1Ca 2 SCHEME 1 Although in the presence of Mg 2ϩ all of the states indicated in Scheme 1 exhibit rather high affinities for ATP (K D in the range 0.5-20 M) (8, 9), only the E1Ca 2 state is primed for transfer of the ␥-phosphoryl group to Asp 351 . As counterions to Ca 2ϩ (3), protons bind to the transport sites in place of Ca 2ϩ , stabilizing the E2 conformation. The fully protonated E2 form (E2H 3 in Scheme 1) is phosphorylated by P i at Asp 351 when the pump works in the reverse mode but cannot be phosphorylated by ATP. Since ATP accelerates P i binding (10) and dephosphorylation (11), modulates P i º HOH exchange (12), and binds fairly tightly to the vanadate complexed E2 form (13), E2H 3 must be able to bind ATP without preventing the access of P i to Asp 351 , suggesting that the ␥-phosphoryl group of the bound ATP is at some distance from Asp 351 in this conformation, in contrast to the E1Ca 2 state. A change in the interaction of bound nucleotide with Asp 351 related to enzyme activation by Ca 2ϩ is clearly demonstrated with TNP-8N 3 -ATP 1 that has

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“…The substrate for the ATPase reaction is MgATP, but Mg# + and ATP can bind and dissociate from the catalytic site independently or as the MgATP complex [27]. Mg# + can bind to preformed E1hCa # :ATP, so that the catalytic Mg# + binding site cannot be ' buried ' underneath the ATP.…”

Section: Scheme 3 Effect Of Ph On the E1/e2 Equilibriummentioning

confidence: 99%

What the structure of a calcium pump tells us about its mechanism

2001

Biochem. J.

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The report of the crystal structure of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca(2+)-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647-655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca(2+) binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane alpha-helix M1, with a Ca(2+) ion passing through the first site to reach the second site. The Ca(2+)-ATPase also contains a pair of binding sites for Ca(2+) that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca(2+) ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca(2+) ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca(2+) ions must involve a significant change in the packing of the transmembrane alpha-helices. The link between the phosphorylation domain and the pair of high-affinity Ca(2+) binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane alpha-helices M6 and M7.

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The binding of ATP and magnesium to the calcium adenosinetriphosphatase of sarcoplasmic reticulum follows a random mechanism

Cited by 16 publications

References 49 publications

Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity between Nucleotide Binding and Phosphorylation Domains

Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity between Nucleotide Binding and Phosphorylation Domains

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

What the structure of a calcium pump tells us about its mechanism

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