Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate. Inhibition by calcium ions

Masuda, Hatisaburo; Meis, Leopoldo de

doi:10.1021/bi00747a006

Cited by 251 publications

(110 citation statements)

References 16 publications

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“…This is in agreement with the observed competitive inhibition by ATP on ezyme phosphorylation with Pi (48). Our experiments suggest that ATP, at 0.1 -1.0 mM concentrations, binds again to the phosphorylated intermediate in exchange for ADP.…”

Section: Why Does Stabilization Of Amppcp-e1-2ca 2+ Require High Ligasupporting

confidence: 92%

Concerted Conformational Effects of Ca²⁺and ATP Are Required for Activation of Sequential Reactions in the Ca²⁺ATPase (SERCA) Catalytic Cycle^,

Inesi

Lewis

et al. 2006

Biochemistry

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We relate solution behavior to the crystal structure of the Ca 2+ ATPase (SERCA). We find that nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the N domain, even in the absence of Ca 2+ and Mg 2+ , or to the closed conformation stabilized by thapsigargin (TG). Why then is Ca 2+ crucial for ATP utilization? The influence of AMPPCP, Ca 2+ and Mg 2+ on proteolytic digestion patterns, interpreted in the light of known crystal structures, indicates that a Ca 2+ dependent conformation of the ATPase headpiece is required for a further transition induced by nucleotide binding. This includes opening of the headpiece, which in turn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chain of bound ATP acquires an extended configuration allowing the γ-phosphate to reach Asp351 to form a complex including Mg 2+ . We demonstrate by Asp351 mutation that this "productive" conformation of the substrate-enzyme complex is unstable due to electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the γ-phosphate yielding the phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with high affinity to the transition state complex, but dissociates with lower affinity as the phosphoenzyme undergoes a further conformational change (i.e., E1P to E2-P transition). Finally, we measured low affinity ATP binding to stable phosphoenzyme analogs, demonstrating that the E1-P to E2-P transition and the enzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.The Ca 2+ ATPase of Sarco-Endoplasmic Reticulum (SERCA) is a membrane bound enzyme that uses ATP as an energy source for Ca 2+ transport. The 100 kD ATPase protein includes ten helical segments partitioned within the membrane bilayer, and a headpiece protruding from the cytosolic membrane surface (1,2). Binding of 2 Ca 2+ is required for enzyme (E) activation, whereby the ATP terminal phosphate is utilized to form a phosphorylated enzyme intermediate (E-P). The bound Ca 2+ then undergoes occlusion and vectorial translocation. The catalytic cycle is completed by hydrolytic cleavage of E-P (3-5)

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Section: Why Does Stabilization Of Amppcp-e1-2ca 2+ Require High Ligasupporting

confidence: 92%

Concerted Conformational Effects of Ca²⁺and ATP Are Required for Activation of Sequential Reactions in the Ca²⁺ATPase (SERCA) Catalytic Cycle^,

Inesi

Lewis

et al. 2006

Biochemistry

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“…Also as observed with TG, ATP strongly enhances PLB cross-linking to SERCA2a in the presence of P i (17) and vanadate (Fig. 10), being able to compete for P i (43) or vanadate binding (19,44) to the Ca 2ϩ -ATPase. Given the reputation of TG as an extremely potent, irreversible inhibitor of the Ca 2ϩ -ATPase (18, 31), we were surprised to discover that TG did not disrupt the ternary complex between the PLB supershifters and E2⅐ATP, even when membranes were preincubated for up to 1 h with greater than stoichiometric concentrations of TG.…”

Section: Tg (M)supporting

confidence: 58%

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

Akin

Chen

Jones

2010

Journal of Biological Chemistry

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Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca 2؉ affinity of SERCA2a by competing for Ca 2؉ binding. showed that PLB bound preferentially to E2 with bound nucleotide, forming a remarkably stable complex that is highly resistant to both thapsigargin and vanadate. In the presence of ATP, N30C-PLB had an affinity for SERCA2a approaching that of vanadate (micromolar), whereas PLB3 and PLB4 had much higher affinities, severalfold greater than even thapsigargin (nanomolar or higher). We conclude that PLB decreases Ca 2؉ binding to SERCA2a by stabilizing a unique E2⅐ATP state that is unable to bind thapsigargin or vanadate.Calcium transport by SERCA2a, the Ca 2ϩ -ATPase in cardiac SR, 2 is regulated by the small inhibitory phosphoprotein PLB. It is generally accepted that PLB inhibits Ca 2ϩ -ATPase activity by decreasing the apparent Ca 2ϩ affinity of the enzyme, with little or no effect on maximal velocity (V max ) measured at saturating Ca 2ϩ concentration (1, 2). PLB inhibition of Ca 2ϩ -ATPase activity is reversed by phosphorylation of PLB at Ser 16 and Thr 17 in response to ␤-adrenergic stimulation, dramatically increasing the rate of Ca 2ϩ uptake into the SR, and enhancing the rates of cardiac relaxation and contraction (1, 2). Yet despite its prominent role in regulating cardiac function, the precise molecular mechanism of PLB inhibition remains unclear. PLB exists as a population of homopentamers and monomers in the SR membrane, the monomer being the active form responsible for Ca 2ϩ -ATPase inhibition (3, 4). Several groups have shown that there is a dynamic equilibrium between PLB pentamers, PLB monomers, and PLB-SERCA heterodimers (5-9). Recent studies with chemical cross-linking have suggested a simple mechanism of PLB inhibition, in which the PLB monomer competes with Ca 2ϩ for binding to SERCA2a by stabilizing a single conformational state of the enzyme (Fig. 1) (7, 10 -12). According to this model, PLB stabilizes the low Ca 2ϩ affinity E2 conformation of the Ca 2ϩ pump and blocks the transition to E1, the conformation required for high-affinity Ca 2ϩ binding and ATP hydrolysis (Fig. 1). Thus SERCA2a with PLB bound cannot bind Ca 2ϩ and is catalytically inactive, and PLB must completely dissociate before the enzyme can transition to E1 and initiate Ca 2ϩ transport. By antagonizing formation of E1, PLB significantly decreases the fraction of Ca 2ϩ pumps available to transport Ca 2ϩ at subsaturating Ca 2ϩ concentration. This is manifested as a decrease in the apparent Ca 2ϩ affinity of the Ca 2ϩ -ATPase, the hallmark of PLB inhibition (1, 2). Ideally, to test the idea that PLB competes with Ca 2ϩ for binding to SERCA2a, Ca 2ϩ binding assays would be used to directly determine whether a population of Ca 2ϩ pumps expressed alone and free from PLB bind Ca 2ϩ with higher affinity than a population of Ca 2ϩ pumps co-expressed with PLB. Unfortunately, acc...

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“…In the Ca2+-ATPase, a magnesium phosphoenzyme intermediate with slowly exchanging Mg2 ' remains after dissociation of ADP [47]; it has been shown to react with ADP rather than Mg2 +-ADP in the reverse reaction [48, 491. The phosphoenzyme can also be formed directly from Mg2+ and phosphate at pH 6 [50]. These results all suggest that on phosphorylation by the ATP the MgZf is transferred along with the y-phosphate to the phosphorylation domain and that its interactions with the nucleotide domain are minimal.…”

Section: + -mentioning

confidence: 81%

The predicted secondary structures of the nucleotide‐binding sites of six cation‐transporting ATPases lead to a probable tertiary fold

Taylor

Green

1989

European Journal of Biochemistry

162

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Six cation-dependent transporting ATPases have homologous sequences in the region assigned by chemical labelling to nucleotide binding. Comparison of the most highly conserved segments with other nucleotide-binding domains showed that the sequences were consistent with a mononucleotide-binding fold and enabled a number of likely folding topologies to be limited to two or three alternatives. One of these possible folds was topologically equivalent to adenylate kinase; this was taken as a model in which the significance of conserved amino acids was investigated. In this model conserved amino acids were grouped around a postulated ATP-binding cleft, satisfactorily accounting for their degree of conservation.The recent determination of the amino acid sequences of six cation-transporting ATPases has shown high similarity (30 -50% identity) in limited regions accounting for about one quarter of the molecule [I]. One of the best defined domains, which follows the central tryptic cleavage site of the Ca2 + -ATPase, is labelled by fluorescein isothiocyanate (FITC) in competition with ATP [2-41. Since erythrosin (tetraiodofluorescein) is known to occupy the adenine pocket of the coenzyme site of lactate dehydrogenase, we can regard FITC as a potential affinity label for the ATP-binding site.The nucleotide domain (98 -180 residues long) forms part of the largest extra-membranous region in all the ATPases (280-430 residues). It is situated between two other conserved domains within this region and separated from them by loops of varying length and sequence. In our preliminary analysis of the secondary structure of two Ca'+-transporting ATPases from sarcoplasmic reticulum [5, 61, we concluded that this region cosisted mainly of alternating p-strands and a-helices and that it probably formed a parallel-stranded psheet structure analogous to that of other nucleotide-binding domains [7]. The domain on the N-terminal side includes an aspartyl residue which is phosphorylated by ATP bound to the nucleotide domain. The segment on the C-terminal side is a short (64 residues) but highly conserved region which, by virtue of its direct connection to the membrane must be close to the N-terminal phosphorylation domain. By analogy with known structures of several kinases [8] we assigned a hinge-I like role to this C-terminal domain.Most nucleotide-binding domains contain parallel psheets which, in spite of marked structural differences, have Correspondence to N. M. Green,

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Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate. Inhibition by calcium ions

Cited by 251 publications

References 16 publications

Concerted Conformational Effects of Ca²⁺and ATP Are Required for Activation of Sequential Reactions in the Ca²⁺ATPase (SERCA) Catalytic Cycle^,

Concerted Conformational Effects of Ca²⁺and ATP Are Required for Activation of Sequential Reactions in the Ca²⁺ATPase (SERCA) Catalytic Cycle^,

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

The predicted secondary structures of the nucleotide‐binding sites of six cation‐transporting ATPases lead to a probable tertiary fold

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Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate. Inhibition by calcium ions

Cited by 251 publications

References 16 publications

Concerted Conformational Effects of Ca2+and ATP Are Required for Activation of Sequential Reactions in the Ca2+ATPase (SERCA) Catalytic Cycle,

Concerted Conformational Effects of Ca2+and ATP Are Required for Activation of Sequential Reactions in the Ca2+ATPase (SERCA) Catalytic Cycle,

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

The predicted secondary structures of the nucleotide‐binding sites of six cation‐transporting ATPases lead to a probable tertiary fold

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Resources

About

Concerted Conformational Effects of Ca²⁺and ATP Are Required for Activation of Sequential Reactions in the Ca²⁺ATPase (SERCA) Catalytic Cycle^,

Concerted Conformational Effects of Ca²⁺and ATP Are Required for Activation of Sequential Reactions in the Ca²⁺ATPase (SERCA) Catalytic Cycle^,