E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

Saffioti, Nicolás A.; Sautu, Marilina de; Ferreira-Gomes, Mariela; Rossi, Rolando; Berlin, Joshua R.; Rossi, Juan Pablo F.C.; Mangialavori, Irene C.

doi:10.1016/j.bbamem.2018.11.001

Cited by 8 publications

(15 citation statements)

References 53 publications

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“…E1P and E2P. Because eosin binds to the ATP binding of the E1 and E2 states and blocks ATP phosphorylation, any investigation of the E1P and E2P states would require phosphorylation by ATP before the addition of eosin or the use of ATP and phosphate analogues [13].…”

Section: Discussionmentioning

confidence: 99%

“…10). Both contain high frequencies of lysine residues, i.e., 13.41% for the Na + ,K + -ATPase and 13.40% for the H + ,K + -ATPase [51]. In contrast, the Nterminus of Homo sapiens sarcoplasmic reticulum Ca 2+ -ATPase (SERCA1, fast twitch skeletal muscle isoform, accession number O14983) only possesses 47 residues prior to the start of the first transmembrane sequence.…”

Section: Discussionmentioning

confidence: 99%

“…1). This probe was first introduced to the field by Skou and Esmann [5,6], but it has since been used in many studies on both the Na + ,K + -ATPase [7][8][9][10][11] and the H + ,K + -ATPase [12] by several research groups and has also been applied in studies of the plasma membrane Ca 2+ -ATPase (PMCA) [13] and the ADP/ATP carrier of mitochondrial membranes [12]. The reason for its wide use is that it is capable of binding to the nucleotide binding site of these enzymes, although in the case of the PMCA it was found that eosin inhibition was not competitive with ATP, suggesting an alternative binding site in the case of this enzyme [15,16].…”

Section: Introductionmentioning

confidence: 99%

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Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

Hossain¹,

Li²,

Zhang³

et al. 2020

Biochimica et Biophysica Acta (BBA) - Biomembranes

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A fluorescence ratiometric method utilising the probe eosin Y is presented for estimating the ATP binding site polarity of P-type ATPases in different conformational states. The method has been calibrated by measurements in a series of alcohols and tested using complexation of eosin Y with methyl-β-cyclodextrin. The results obtained with the Na + ,K +-, H + ,K +-and sarcoplasmic reticulum Ca 2+-ATPases indicate that the ATP binding site, to which eosin is known to bind, is significantly more polar in the case of the Na + ,K +-and H + ,K +-ATPases compared to the Ca 2+-ATPase. This result was found to be consistent with docking calculations of eosin with the E2 conformational state of the Na + ,K +-ATPase and the Ca 2+-ATPase. Fluorescence experiments showed that eosin binds significantly more strongly to the E1 conformation of the Na + ,K +-ATPase than the E2 conformation, but in the case of the Ca 2+-ATPase both fluorescence experiments and docking calculations showed no significant difference in binding affinity between the two conformations. This result could be due to the fact that, in contrast to the Na + ,K +-and H + ,K +-ATPases, the E2-E1 transition of the Ca 2+-ATPase does not involve the movement of a lysine-rich N-terminal tail which may affect the overall enzyme conformation. Consistent with this hypothesis, the eosin affinity of the E1 conformation of the Na + ,K +-ATPase was significantly reduced after N-terminal truncation. It is suggested that changes in conformational entropy of the N-terminal tail of the Na + , K +-and the H + ,K +-ATPases during the E2-E1 transition could affect the thermodynamic stability of the E1 conformation and hence its ATP binding affinity.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

Hossain¹,

Li²,

Zhang³

et al. 2020

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…In the case of acidic phospholipid activation, 24 μg/ml of brain extract (Type I Folch Fraction I from bovine brain containing approximately 10% phosphatidylinositol, 50% phosphatidylserine, and other lipids) were added to the medium (26). In Figures 1 and 2A-D, the effect of metal fluoride complexes on Ca 2+ -ATPase activity was assessed as described previously by Saffioti et al (2019) (15). AlF and BeF were formed by combining 1.5 mM NaF and the AlCl3 or BeCl2 concentration indicated in the figures.…”

Section: Methodsmentioning

confidence: 99%

“…In the absence of Ca 2+ , BeF3 -, AlF4and MgF4 2-bind to the Asp 351 residue of SERCA mimicking the phosphoryl group in the E2P ground, E2P phosphoryl transition and E2•Pi product states of the E2P dephosphorylation reaction, respectively (E2P-like states) (11)(12)(13)(14). In PMCA, we have demonstrated that metal fluoride complexes (MeF) also stabilize analogues of the phosphorylated state, and characterized the kinetics of their formation (15).…”

Section: Introductionmentioning

confidence: 99%

Conformational changes during the reaction cycle of Plasma Membrane Ca2+-ATPase in the autoinhibited and activated states

Saffioti

Sautu

Riesco

et al. 2021

Biochemical Journal

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Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity.  Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2∙Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

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Novel scaffolds targeting Mycobacterium tuberculosis plasma membrane Ca2+ transporter CtpF by structure-based strategy

Varon

Santos

López‐Vallejo

et al. 2023

Bioorganic Chemistry

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E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

Cited by 8 publications

References 53 publications

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

Conformational changes during the reaction cycle of Plasma Membrane Ca2+-ATPase in the autoinhibited and activated states

Novel scaffolds targeting Mycobacterium tuberculosis plasma membrane Ca2+ transporter CtpF by structure-based strategy

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