Importance of Thr-353 of the Conserved Phosphorylation Loop of the Sarcoplasmic Reticulum Ca2+-ATPase in MgATP Binding and Catalytic Activity

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Residues in conserved motifs 625 The sarcoplasmic reticulum Ca 2ϩ -ATPase 1 from rabbit muscle couples the hydrolysis of ATP to the transport of Ca 2ϩ to the reticular lumen, and the lowering of the sarcoplasmic Ca 2ϩ 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 Ca 2ϩ binding in the E1 form, and hydrolysis of the aspartyl phosphoryl bond occurs in the E2P phosphoenzyme conformation following Ca 2ϩ translocation across the membrane. Two atomic structures of the protein have been elucidated by x-ray crystallography, one in the presence of Ca 2ϩ (designated E1(Ca 2 )) and another in the absence of Ca 2ϩ and presence of the specific inhibitor thapsigargin (E2(TG)) (1, 2). 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(Ca 2 ) and closer together, forming a more compact entity, in E2(TG). For ATP-dependent phosphorylation of Asp 351 to occur in the Ca 2ϩ bound E1 state, domain N must close over the phosphorylation site, such that the nucleotide site is brought close to Asp 351 . 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 625 TGD, 676 FARXXPXXK, and 701 TGDGVND that surround the phosphorylation loop 351 DKTGTLT. 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 Ca 2ϩ -ATPase and related pumps (for review, see Ref.3). Most of them recur at the active site of a large class of soluble phosphohydrolases and phosphotransferases believed to have a similar reaction mechanism (4).

mentioning

confidence: 51%

“…The reaction was started by the addition of ATP to the Ca 2ϩ -bound Ca 2ϩ -ATPase. Other phosphorylation and dephosphorylation experiments were carried out by manual mixing techniques (7). Acid quenching was performed with 0.5-2 volumes of 25% (w/v) trichloroacetic acid containing 100 mM H 3 PO 4 .…”

Section: Methodsmentioning

confidence: 99%

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

McIntosh

Clausen

Woolley

et al. 2004

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“…Closure of the cleft between N-and P-domain is thought to occur to bring ATP close to Asp 351 upon nucleotide binding (20,21). In line with that, mutations at Asp 351 , Lys 352 (25), and Thr 353 (26) alter the affinity of ATP to the ATPase.…”

mentioning

confidence: 79%

“…If an interaction between nucleotide and ATPase had only local effects on the protein structure, a weakened interaction would selectively reduce the amplitude of difference bands associated with that conformational change, but not of all of the bands as observed here. Particularly interesting is that functional groups of ATP, which interact with different domains of the protein, produce the same type of conformational change: the amino function is thought to interact with the N-domain (19,24,46) and the ␥-phosphate with the P-domain (25,26,46). Despite that, the absence of the ␥-phosphate in ADP (28) or of the adenine amino group in ITP both reduce the amplitude of the same bands.…”

Section: Discussionmentioning

confidence: 99%

Mapping Interactions between the Ca2+-ATPase and Its Substrate ATP with Infrared Spectroscopy

Liu

Barth

2003

Infrared spectroscopy has been used to map substrate-protein interactions: the conformational changes of the sarcoplasmic reticulum Ca 2؉ -ATPase upon nucleotide binding and ATPase phosphorylation were monitored using the substrate ATP and ATP analogues (2 -deoxy-ATP, 3 -deoxy-ATP, and inosine 5 -triphosphate), which were modified at specific functional groups of the substrate. Modifications to the 2 -OH, the 3 -OH, and the amino group of adenine reduce the extent of bindinginduced conformational change of the ATPase, with particularly strong effects observed for the latter two. This demonstrates the structural sensitivity of the nucleotide-ATPase complex to individual interactions between nucleotide and ATPase. All groups studied are important for binding and interactions of a given ligand group with the ATPase depend on interactions of other ligand groups.Phosphorylation of the ATPase was observed for ITP and 2 -deoxy-ATP, but not for 3 -deoxy-ATP. There is no direct link between the extent of conformational change upon nucleotide binding and the rate of phosphorylation showing that the full extent of the ATP-induced conformational change is not mandatory for phosphorylation. As observed for the nucleotide-ATPase complex, the conformation of the first phosphorylated ATPase intermediate E1PCa 2 also depends on the nucleotide, indicating that ATPase states have a less uniform conformation than previously anticipated.Ligand binding to proteins controls vast numbers of cellular processes and has attracted great scientific and economic interest. Protein and ligand flexibility are important determinants of the interaction and often lead to ligand binding modes that are not anticipated from structures obtained with other ligands. To these "failure(s) of the rigid receptor hypothesis" (1) is added here an impressive example: induced-fit binding of nucleotides to the Ca 2ϩ -ATPase. This finding stems from a systematic mapping of substrate-protein interactions with infrared (IR) 1 spectroscopy. New approaches like this are welcome in the field of ligand-protein recognition, since the most informative techniques, NMR and x-ray crystallography, are laborious and problematic for some systems. Methods like fluorescence and luminescence that require less expenditure also provide less molecular information. We expect that this technology gap will be bridged by IR spectroscopy.IR spectroscopy, one of the methods of vibrational spectroscopy, provides direct information on the molecular level, is cost-effective, and can be universally applied from small soluble proteins to large membrane proteins under near-physiological conditions. Work summarized in recent reviews (2-5) has shown that the vibrational spectrum changes characteristically when a ligand binds to a protein. This provides a direct observation of ligand binding: no marker compound has to be introduced to report the binding process, as with many other methods. Previous work has mostly focused on individual interactions between a ligand and a protein by monitoring the ...

“…Most published studies of purified enzyme in aqueous solution support this interpretation (3)(4)(5)(6)(7). However, a study in an organic solventwater mixture concluded that the true substrate is Mg⅐P i (8), and recently the equation for the half-maximum Mg⅐P i concentration was used to interpret an increased half-maximum P i concentration when the upstream threonine in a signature amino acid sequence for P-type pumps (DKTGT) was changed to serine (9). The interpretation given to most studies of Na,KATPase is also that Mg 2ϩ and P i bind randomly (10 -12).…”

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confidence: 99%

Evidence Calcium Pump Binds Magnesium before Inorganic Phosphate

Nagy

Kane

Tran

et al. 2005

Calcium pump-catalyzed18 O exchange between inorganic phosphate and water was studied to test the hypothesis that all P-type pumps bind Mg 2؉ before P i and validate utilization of the rate equation for ordered binding to interpret differences between site-directed mutants and wild-type enzyme. The results were remarkably similar to those obtained earlier with sodium pump (Kasho, V. N., Stengelin, M., Smirnova, I. N., and Faller, L. D. (1997) Biochemistry 36, 8045-8052). The equation for ordered binding of Mg 2؉ before P i fit the data best with only a slight chance (0.6%) of P i binding to apoenzyme. Therefore, P i is the substrate, and Mg 2؉ is an obligatory cofactor. The intrinsic Mg 2؉ dissociation constant from metalloenzyme (K M ‫؍‬ 3.5 ؎ 0.3 mM) was experimentally indistinguishable from the sodium pump value. However, the half-maximal concentration for P i binding to metalloenzyme (K P ‫؍‬ 6.3 ؎ 0.6 mM) was significantly higher (ϳ6-fold), and the probability of calcium pump forming phosphoenzyme from bound P i (P c ‫؍‬ 0.04 ؎ 0.03) was significantly lower (ϳ6-fold) than for the sodium pump. From estimates of the rate constants for phosphorylation and dephosphorylation, the calcium pump appears to catalyze phosphoryl group transfer less efficiently than the sodium pump. Ordered binding of Mg 2؉ before P i implies that both calcium pump and sodium pump form a ternary enzyme⅐metal⅐phos-phate complex, consistent with molecular structures of other haloacid dehalogenase superfamily members that were crystallized with Mg 2؉ and phosphate, or a phosphate analogue, bound.The calcium and sodium pumps are classified as P-type, 1 because the energy required for generating ion gradients across cell membranes is derived from ATP by catalyzing hydrolysis in two, Mg 2ϩ -dependent steps (1). The ␥-phosphoryl group is initially transferred to an aspartyl side chain of the protein in one conformation (E 1 ) forming a covalent phosphoenzyme intermediate. Hydrolysis is completed after a conformational change by transferring the phosphoryl group to water. The second step can be reversed by reacting the second conformation (E 2 ), with P i and can be followed either by radioactive 32 P incorporation into the enzyme, or by stable oxygen isotope ( 18 O) exchange between P i and H 2 O. Phosphorylation by P i is a partial reaction that can be uncoupled from other reactions in the pump cycle experimentally, so that the mechanism is simple enough for derivation of the rate equation. Therefore, measurements combining either 32 P incorporation or 18 O exchange with site-directed mutagenesis are potentially a powerful way of identifying amino acids essential for catalyzing phosphoryl group transfer and of learning their function from estimates of intrinsic constants for interaction of reactants with the enzyme.The problem considered in this report is the mechanism of Mg 2ϩ -dependent phosphorylation by P i . The solution is important because the interpretation of observed differences between mutants and wild-type enzyme depends upon the...